THE IMPACT OF INCREASING CARBOHYDRATE INTAKE DOSES ON EXOGENOUS CARBOHYDRATE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE PERFORMANCE Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. ____________________________________________________ JohnEric William Smith _________________________________ _________________________________ L. Bruce Gladden David D. Pascoe, Chair Professor Professor Kinesiology Kinesiology _________________________________ _________________________________ G. Dennis Wilson Jeffrey J. Zachwieja Professor Emeritus Adjunct Professor Kinesiology Gatorade Sports Science Institute _________________________________ Joe F. Pittman Interim Dean Graduate School THE IMPACT OF INCREASING CARBOHYDRATE INTAKE DOSES ON EXOGENOUS CARBOHYDRATE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE PERFORMANCE JohnEric William Smith A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, AL May 10, 2008 iii THE IMPACT OF INCREASING CARBOHYDRATE INTAKE DOSES ON EXOGENOUS CARBOHYDRATE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE PERFORMANCE JohnEric William Smith Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. ______________________________ Signature of Author ______________________________ Date of Graduation iv VITA JohnEric W. Smith, son of Kenneth Rodger and Gail Underwood Smith, was born December 15, 1976, in LaGrange, Georgia. He graduated from Central High School, Carrollton, Georgia, in 1995. JohnEric married Kimberly Odum, daughter of James and Jeanie Odum, on June 10, 2000. He graduated with a Bachelor of Science degree in Exercise Science in August 2000. In May of 2003, JohnEric received his Masters of Science degree in Exercise Science and began his doctoral studies at Auburn University. In November 2004, JohnEric accepted a scientist position at the Gatorade Sports Science Institute. v DISSERTATION ABSTRACT THE IMPACT OF INCREASING CARBOHYDRATE INTAKE DOSES ON EXOGENOUS CARBOHYDRATE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE PERFORMANCE JohnEric William Smith Doctor of Philosophy, May 10, 2008 (M.S., Auburn University, 2003) (B.S., Auburn University, 2000) 142 Typed Pages Directed by David D. Pascoe This study assessed: (1) the impact of increasing carbohydrate dosages on carbohydrate oxidation and (2) the impact of increasing carbohydrate dosages on exercise performance. Twelve male cyclists/triathletes ingested a placebo or glucose drinks delivering 15, 30, and 60 g?hr -1 during 120 minutes of constant load cycling at ~75% VO 2 PEAK . Glucose drinks were extrinsically labeled with 1.8 mg?g -1 U- 13 C-glucose and a 20- km time-trial followed each constant load ride. Blood glucose and insulin were highest when ingesting 60 g?hr -1 while free fatty acids were the lowest. Insulin and free fatty acid responses for placebo and the 15 g?hr -1 trial were virtually identical. Exogenous glucose oxidation rates during the last 30 minutes of the constant load cycling (mean ? vi SE) were 0.26 ? 0.05, 0.44 ? 0.04 and 0.66 ? 0.07 g?min -1 for 15, 30 and 60 g?hr -1 ingestion rates, each being significantly different from each other (p ? 0.05). Liver glucose oxidation rate was highest when consuming 15 g?hr -1 (0.63 ? 0.13 g?min -1 ) followed by 30 g?hr -1 (0.51 ? 0.12 g?min -1 ) and 60 g?hr -1 (0.42 ? 0.08 g?min -1 ), all significantly different from one another at a p ? 0.05 level. There was also significant reduction in muscle glycogen oxidation during the last hour of the 2-hour constant load ride with no significant differences between the 15, 30, and 60 trials. Relative to placebo, glucose ingestion improved time-trial performance (p ? 0.05) with no statistical difference between glucose doses. These findings suggest the ingestion of glucose at increasing rates, between 0 g?hr -1 and 60 g?hr -1 , reduce the demand on liver glucose and carbohydrate ingestion rates as low as 15 g?hr -1 can improve cycling time-trial performance. vii ACKNOWLEDGEMENTS The author would like to express his love and appreciation for his wife. She supported, encouraged, and understood the time required to complete his studies. The author also would like to thank his parents, grandparents, and brother for pushing him to finish his degree. Thanks go to my committee members for their valuable input. Thanks also go to the Gatorade Sports Science Institute staff for their tutelage and support. Special thanks go to Bob Murray and Jeff Zachwieja for their encouragement and providing the author the opportunity to finish his degree while also beginning his career. Thanks most also be given to Franois Peronnet, Denis Massicotte, and Carole Lavoie for teaching me the isotope methodology used in this project. A very special thanks is given to David Pascoe for his mentorship and friendship through the authors graduate studies. Above all, the author would like to thank God for all of the opportunities and blessings that he has provided. viii Style manual or journal used Journal of Applied Physiology Computer Software used Microsoft Word for Windows XP ix TABLE OF CONTENTS LIST OF FIGURES .............................................................................................................x I. INTRODUCTION ..................................................................................................1 II. REVIEW OF LITERATURE ..................................................................................8 III. METHODS. .......................................................................................................... 17 IV. RESULTS ..............................................................................................................25 V. IMPACT OF LOW DOSE GLUCOSE INGESTION ON EXOGENOUS GLUCOSE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE .......................................................................47 VI. SUMMARY...........................................................................................................88 REFERENCES ..................................................................................................................90 APPENDICES .................................................................................................................112 APPENDIX A: Subject Informed Consent ........................................................113 APPENDIX B: Data Collection Sheet................................................................123 x LIST OF FIGURES IV. 1. ORDER OF TEST MEASURES.......................................................................29 IV. 2. 20-KILOMETER COURSE PROFILE.............................................................30 IV. 3. HEART RATE DURING 2-HOUR CONSTANT LOAD RIDE ..................................................................................................................31 IV. 4. RATINGS OF PERCEIVED EXERTION DURING THE 2-HOUR CONSTANT LOAD RIDE ................................................................32 IV. 5. V & O 2 DURING EXERCISE ...............................................................................33 IV. 6. RESPIRATORY EXCHANGE RATIO DURING EXERCISE ........................................................................................................34 IV. 7. 13 C/C RATIO IN EXPIRED AIR DURING EXERCISE ................................35 IV. 8. CARBOHYDRATE OXIDATION DURING EXERCISE ..............................36 IV. 9. FAT OXIDATION DURING EXERCISE........................................................37 IV. 10. BLOOD GLUCOSE RESPONSE DURING EXERCISE ................................38 IV. 11. INSULIN RESPONSE DURING EXERCISE..................................................39 IV. 12. FREE FATTY ACID RESPONSE DURING EXERCISE ...............................40 IV. 13. LACTATE DURING 2-HOUR CONSTANT LOAD EXERCISE ........................................................................................................41 IV. 14. CORTISOL RESPONSE DURING EXERCISE ..............................................42 xi IV. 15. PERCENT PLASMA GLUCOSE COMING FROM AN EXOGENOUS SOURCE ..................................................................................43 IV. 16. SOURCE OF OXIDIZED CARBOHYDRATE DURING THE LAST HOUR OF EXERCISE WHEN CONSUMING GLUCOSE AT 15, 30, AND 60 g?h -1 ......................................44 IV. 17. CHANGE IN 20-KM TIME TRIAL COMPLETION TIME IN RELATION TO PLA.........................................................................45 IV. 18. CHANGE IN 20-KM TIME TRIAL AVERAGE WATTAGE IN RELATION TO PLA...............................................................46 V. 1. ORDER OF TEST MEASURES .......................................................................75 V. 2. 20-KILOMETER COURSE PROFILE .............................................................76 V. 3. V & O 2 DURING EXERCISE ...............................................................................77 V. 4. RESPIRATORY EXCHANGE RATIO DURING EXERCISE ........................................................................................................78 V. 5. 13 C/C RATIO IN EXPIRED AIR DURING EXERCISE .................................79 V. 6. CARBOHYDRATE OXIDATION DURING EXERCISE...............................80 V. 7. FAT OXIDATION DURING EXERCISE........................................................81 V. 8. BLOOD GLUCOSE RESPONSE DURING EXERCISE.................................82 V. 9. INSULIN RESPONSE DURING EXERCISE..................................................83 V. 10. FREE FATTY ACID RESPONSE DURING EXERCISE ...............................84 V. 11. PERCENT PLASMA GLUCOSE COMING FROM AN EXOGENOUS SOURCE ..................................................................................85 xii V. 12. SOURCE OF OXIDIZED CARBOHYDRATE DURING THE LAST HOUR OF EXERCISE WHEN CONSUMING GLUCOSE AT 15, 30, AND 60 g?h -1 ......................................86 V. 13. CHANGE IN 20-KM TIME TRIAL COMPLETION TIME IN RELATION TO PLA.........................................................................87 1 I. INTRODUCTION Fatigue during prolonged exercise has often be associated with hypoglycemia and depletion of muscle glycogen. There is a great deal of evidence demonstrating the ergogenic effect of consuming carbohydrate before and during endurance exercise (2; 12; 20; 21; 29; 31; 42; 43; 56; 121; 130; 142; 144). The American College of Sports Medicine (ACSM) and the National Athletic Trainers Association (NATA) both have fluid replacement position stands that promote the benefit of carbohydrate intake during exercise. The position stands both suggest carbohydrate ingestion rates of 30-60 g?hr -1 (18; 123). These positions and experts? findings have led to the use of carbohydrate drinks during prolonged exercise as common practice. A great deal of research has been conducted to show carbohydrate ingestion rate that optimizes exercise performance and substrate utilization (20; 29; 31; 34; 49; 61; 70; 125). Results indicate that when carbohydrate is consumed during exercise, time to exhaustion is prolonged (12; 20; 21; 29; 31; 42; 43; 56; 121; 130; 142; 143). Carbohydrate intake during exercise has also been shown to reduce time required to complete a set distance or work volume (2; 4; 6; 8; 92; 93; 95; 97). Improvements in exercise performance have been reported with carbohydrate intakes during exercise ranging from 18 to 180 g?h -1 (31; 34; 42; 49; 89; 90; 92; 95; 143). 2 The improvements in performance experienced with carbohydrate ingestion have been attributed to the maintenance of carbohydrate oxidation late during exercise (14; 21; 29; 136; 142). Carbohydrate oxidation is maintained through the oxidation of exogenous carbohydrate sources (85; 98; 115), the maintenance of blood glucose,(4; 5; 14; 21; 29; 49; 57; 136) and the sparing of endogenous carbohydrate (80; 98; 115). Most research suggests that liver glycogen is spared through the ingestion of carbohydrate prior to and during exercise (5; 49; 55; 57; 70; 85). The sparing of muscle glycogen is not typically reported during cycling exercise when carbohydrate is ingested (14; 20; 29; 31; 42; 43; 89; 125). Many studies have tested carbohydrate ingestion rates above those recommended by the ACSM and NATA position stands in hopes of optimizing exercise performance and exploring the possibility of a carbohydrate/performance dose response. These studies have failed to demonstrate greater enhancements in performance with carbohydrate ingestion rates above ACSM and NATA recommended ingestion rates compared to the recommended rate range (33; 89; 93). Much less research has been conducted to determine if there is a minimum level of carbohydrate intake needed for improved exercise performance and if there is a dose response to varying carbohydrate levels below what is typically found in sports drinks (17; 42; 138; 143). While it might be logical that providing increased levels of carbohydrate will provide additional energy for exercise, research has shown that high carbohydrate ingestion rates have been associated with an increased incidence of stomach discomfort and stomach upset which may result in diminished exercise performance (114; 119; 135). Research has also demonstrated that when only glucose is ingested the body is able to 3 utilize it at a rate of about 1 g?min -1 (14; 64; 70; 80; 136). When multiple carbohydrates (i.e. glucose and fructose) are consumed, glucose oxidation rates have been reported to be up to and above 1.5 g?min -1 (14; 61; 64; 69; 70; 80; 136). This study examined the physiological and exercise performance responses of cyclists during a 2-hour constant load ride followed by a simulated 20-km time trial to determine the impact of carbohydrate delivery rates below what is typically recommended (60 g?hr -1 ) for prolonged exercise. Participants completed four trials including a trial with no carbohydrate and three trials where carbohydrate was ingested in a drink at a rate of 15 g?h -1 , 30 g?h -1 , and 60 g?h -1 . 4 OPERATIONAL DEFINITIONS 13 C ? an isotope of carbon containing an additional neutron with a molecular weight of 13 CompuTrainer? ? a bicycle trainer and computer system that allows cyclists to perform cycling exercise while simulating the variations in work that one would experience riding on the road Endogenous Carbohydrate ? carbohydrate that is consumed and stored within the body outside of a testing period Endogenous Carbohydrate Oxidation ? a process in which endogenous carbohydrate stored within the body, prior to the testing period, is being metabolized for energy Exogenous Carbohydrate ? carbohydrate that is consumed during a testing period Exogenous Carbohydrate Oxidation ? a process in which exogenous carbohydrate being ingested is being metabolized for energy Glucose Oxidation ? the metabolism of glucose for energy Pee Dee Bellemnitella (PDB) ? a description of the ratio of 13 C/ 12 C compared to the Chicago Standard Plasma ? Fluid portion of blood remaining after the removal of red blood cells Respiratory Exchange Ratio ? the ratio of V & CO 2 and V & O 2 , often used to describe carbohydrate and fat contribution to energy Serum ? Fluid portion of blood remaining after the removal of red blood cells and clotting factors Stable Isotope ? an isotope of an atomic molecule that is not radioactive 5 Urine Specific Gravity ? a measure of particle concentration in urine as compared to distilled water V & O 2 PEAK ? the highest oxygen uptake measured during a graded exercise test 6 The working hypotheses for this investigation are as follows: Exogenous Carbohydrate Oxidation: H O : The null hypothesis states there is no difference in exogenous carbohydrate oxidation as carbohydrate ingestion rate increases. H A : The alternative hypothesis states there is an increase in exogenous carbohydrate oxidation as carbohydrate ingestion rate increases. Exogenous carbohydrate oxidation is calculated from the change in 13 C/ 12 C ratio in expired CO 2 with the ingestion of 13 C labeled glucose. Endogenous Carbohydrate Oxidation: H O : The null hypothesis states there is no difference in endogenous carbohydrate oxidation as carbohydrate ingestion rate increases. H A : The alternative hypothesis states there is a decrease in endogenous carbohydrate oxidation as carbohydrate ingestion rate increases. Endogenous carbohydrate oxidation is calculated by subtracting exogenous carbohydrate oxidation rate from total carbohydrate oxidation. Time Trial Performance: H O : The null hypothesis states there is no difference in time required to complete a 20-km time-trial following 2-hours of constant load cycling when carbohydrate ingestion rate is increased. 7 H A : The alternative hypothesis states there is a decrease in the time required to complete a 20-km time-trial following 2-hours of constant load cycling when carbohydrate ingestion rate is increased. 20-km time-trial completion time is measured by the CompuTrainer computer program. Delimitations 1. Only experienced male cyclists and triathletes were included as subjects 2. Only 12 subjects were tested 3. Subjects performed the 20-km time-trial after 2-hours of constant load cycling exercise 4. Subjects performed the 20-km time-trial on a computer simulated cycling course 5. Subjects began the 2-hour constant load cycling exercise after a 10-hour fast Limitations 1. Endogenous carbohydrate oxidation was not directly measured 2. Subjects were not allowed to drink ad libitum 8 II. REVIEW OF LITERATURE Fatigue during prolonged exercise has been associated with hypoglycemia and depletions of muscle glycogen (20; 29; 53; 129). There is a great deal of evidence demonstrating the ergogenic effect of consuming carbohydrate before and during endurance exercise (2; 12; 14; 20; 21; 29; 31; 42; 43; 56; 121; 130; 142; 143). Sport drinks provide a good avenue to replenish fluids lost during exercise as well as an opportunity to take in an exogenous source of energy (20; 89). The American College of Sports Medicine (ACSM) and the National Athletic Trainers Association (NATA) both have fluid replacement position stands that promote the benefit of carbohydrate intake during exercise (18; 123). Both position stands recommend carbohydrate ingestion rates of 30-60 g?hr -1 . These research studies and position statements have led to the common use of carbohydrate supplementation during prolonged exercise. A practical hypothesis would suggest greater carbohydrate ingestion rates would enhance exercise performance. The ergogenic effect of carbohydrate feeding is thought to be partially related to the maintenance of plasma glucose levels and to an increased contribution of glucose as substrate for working muscle (4; 14; 20; 21; 29; 89; 115; 130; 136). During prolonged exercise blood glucose and intramuscular glycogen are the two major sources of carbohydrate utilization by the active muscles (40). A continuous decline in plasma glucose can be observed during exercise when not consuming exogenous energy. This decline demonstrates that hepatic glucose output cannot match glucose uptake by 9 exercising muscles (93). Carbohydrate oxidation is maintained at higher rates when circulating blood glucose is better maintained (136). Massicotte et al (1986) found that blood glucose is maintained with glucose and fructose ingestion (80). Many cycling studies have found that when carbohydrate is ingested there is a reduction in the decline in blood glucose levels and total carbohydrate oxidation observed during prolonged exercise when consuming a placebo (12; 22; 29; 31; 89). Murray et al (1991) found that the ingestion of carbohydrate during 2-hours of cycling exercise helped maintain blood glucose levels and resulted in improved subsequent exercise performance (93). Tsintzas et al (1998) suggested that the supplementation of carbohydrate will delay fatigue by preventing the reduction in blood glucose and providing an immediately usable fuel source to the exercising muscles (129). Massicotte and colleagues (1986) also reported elevated insulin levels following glucose ingestion as compared to water and fructose (80). In 1985, Koivisto reported that glucose ingestion can lead to a seven fold rise in plasma insulin levels (73). The increase of serum insulin concentrations will promote an increase in the amount of glucose uptake by the muscle (85; 143). Nicholas (1999) suggested the increased glucose uptake by the exercising muscle would reduce the depletion of muscle glycogen stores by providing an alternative energy source (98). Increased plasma glucose, as a result of carbohydrate ingestion, reduces the rise in plasma free fatty acids (29; 31). Increases in plasma insulin levels also result in a decline circulating free fatty acid levels (140). This suggests that with increased carbohydrate availability the need to utilize fat for energy to sustain performance is reduced. Massicotte et al (1986) reported that the ingestion of both exogenous glucose and 10 fructose reduced endogenous carbohydrate and fat utilization as compared to placebo. However, fat utilization was significantly higher when fructose was ingested as compared to ingesting glucose (80). Muscle glycogen is a crucial energy source to prevent exhaustion during prolonged exercise (10; 11; 54). Both exercise duration and intensity affect the rate of glycogen breakdown during exercise (10; 11; 72). Research shows significant muscle glycogen depletion occurs only after 90-120 minutes of exercise at ~70% V & O 2max (29). It has been suggested that ingested carbohydrate can be used for energy protecting intramuscular glycogen stores which can then be used later during exercise (5; 9; 11; 12; 25; 38; 46; 48-50; 75; 78-81; 98; 104; 108; 110; 113; 124; 126; 130; 131; 143). Researchers suggest that carbohydrate ingestion during exercise does not result in sparing of muscle glycogen stores (14; 20; 29; 31; 42; 43; 73; 89; 100; 125). Most of the experiments that have demonstrated muscle glycogen sparing have used muscle biopsy techniques. A reason for the different findings may be the single muscle group analysis with the biopsy technique compared to the total body analysis with the tracer technique. Another possible explanation for the differences in metabolism between the studies may be accounted for by the exercise form chosen for the studies (130). These differences may help account for the differences found in the literature. While there is debate concerning muscle glycogen sparing, research demonstrates hepatic glucose output is reduced with carbohydrate ingestion (14; 70; 85). Exogenous carbohydrate appears to supplement hepatic glucose production and maintain blood glucose levels (5; 29; 49; 57; 136). With exogenous glucose aiding in the maintenance of plasma glucose less demand is placed on the liver for glucose production (14; 70; 85). 11 With available carbohydrate stores diminishing there is a reduction in carbohydrate oxidation during prolonged exercise. Carbohydrate oxidation can be maintained during the latter stages of prolonged exercise with carbohydrate feeding (14; 21; 29; 142). McConnell et al (1994) suggested that when glycogen stores are depleted in adults the primary source of energy in prolonged exercise comes from exogenous carbohydrate sources (85). In 1998, Sugiura et al reported that carbohydrate oxidation was lower in placebo trials as compared to trials when subjects received either glucose or fructose (127). Depending on the type of carbohydrate ingested benefits can vary. Massicotte et al (1986) reported that 75% of ingested glucose was oxidized over a 3-hour exercise period whereas only 56% of ingested fructose was oxidized (80). However, there is no difference in carbohydrate delivery or oxidation when glucose polymer is ingested as compared to the ingestion of a simple glucose solution (52; 81). Hawley et al (1992) found that there was no difference in carbohydrate oxidation rates when either glucose or maltose was ingested (52). Wagenmakers et al (1993) found no difference in the oxidation rates of sucrose and maltodextrin (136). In a series of papers by Jentjens and Jeukendrup, it was found that ingesting multiple forms of carbohydrate during exercise resulted in a significantly higher oxidation rate as compared to ingesting a single carbohydrate form (61; 64; 69). The amount and type of carbohydrate ingested will also play a part in its availability to muscle as fuel. For ingested carbohydrate to be utilized by the exercising muscle, it must first be emptied from the stomach and absorbed by the intestines. Early research suggested that beverages designed to rehydrate should not contain more 2.5% 12 carbohydrate concentrations levels because carbohydrate beverages emptied from the stomach more slowly than water (7; 27; 30; 44). Later research determined that carbohydrate (glucose, fructose, sucrose, and maltodextrin) concentrations between 5 and 7.5% emptied from the stomach at a rate no different than water (90; 96). Other researchers have reported that carbohydrate concentrations in excess of 6% can reduce the rate of gastric emptying (114; 116; 135). Caloric content has been determined to be the primary determinant of gastric emptying (86; 91). Once the ingested carbohydrate reaches the intestine, the type of carbohydrate can impact the rate absorption. Carbohydrate concentrations in excess of 6% can slow the rate of absorption (119). Glucose is absorbed in the intestine by a sodium dependent glucose transporter (SGLT 1) in the brush border membrane and paracellular absorption (41; 116). Fructose is reportedly absorbed via facilitated diffusion and GLUT 5 transporters (15; 41; 47; 117). The rate at which fructose is absorbed in humans is slower than that seen with glucose (112). Due to the delayed absorption rate with fructose, ingestion can lead to gastric discomfort and distress (94; 127). Not all ingested carbohydrate is oxidized for energy. When a single form of carbohydrate is ingested the percentage being oxidized ranges from 32-80% (61; 64; 69; 70; 80; 136). When multiple carbohydrate forms are ingested the percentage that is being oxidized ranges from 55-93% (61; 64; 69; 136). Jeukendrup (1999) and Jentjens (2004) demonstrated that increased ingestion rates resulted in lower percentages of the ingested carbohydrate being oxidized (61; 69). Questions arise as to the reason all of the ingested carbohydrate is not oxidized even when carbohydrate ingestion rate is low. It has been reported that most carbohydrate ingested is emptied from the stomach, meaning gastric 13 emptying is not the rate limiting step for exogenous carbohydrate oxidation (114). It has been suggested that some of the carbohydrate that is ingested may remain in the intestines or may go to the liver or inactive muscles to be stored (70). Research has shown that there is a threshold to carbohydrate intake that when exceeded does not improve endogenous carbohydrate sparing or performance. Wagenmakers et al (1993) found that carbohydrate intakes above 74 g?hr -1 did not improve carbohydrate oxidation or endogenous carbohydrate sparing (136). Jentjens et al reported that exogenous glucose oxidation rates do not increase when glucose intake is increased from 1.2 g?min -1 to 1.8 g?min -1 (61; 136). Methodology has been developed and tested demonstrating the ability to use isotopes of carbon to non-invasively analyze the oxidation rates of exogenous carbohydrate and estimate endogenous substrate utilization during exercise (35; 48; 50; 52; 58; 61; 64; 69; 74; 78-82; 102; 104-106; 108-110; 113; 115; 122; 136). This type of research has demonstrated that various forms of carbohydrate consumed immediately prior to and during exercise is readily used for energy (61; 64; 80; 81; 104; 108). The rate at which glucose enters the systemic system seems to be the limiting factor for exogenous carbohydrate oxidation (70). When a single type of carbohydrate is ingested peak oxidation rates are approximately 1 g?min -1 (51; 65; 70; 109; 120; 136). This is likely the result of intestinal SGLT 1 glucose transporters being saturated. It has been reported that SGLT 1 may be saturated at 1.0 g?min -1 (111). Exogenous oxidation rates have been reported to exceed 1.2 g?min -1 when multiple forms of carbohydrate are ingested at high rates (61; 64; 69). 14 The increase in exogenous oxidation rate may be the result of the ability to use both glucose and fructose intestinal transporters (61). When using carbon isotope techniques several assumptions are made and need to be considered before making conclusions. The computation of the oxidation rate of exogenous glucose is made assuming that, in response to exercise, 13 C is not irreversibly lost in pools of the tricarboxylic acid cycle intermediates and/or bicarbonates and that 13 CO 2 recovery in expired gases is complete or almost complete (23; 76). Since some of the ingested 13 C is going into the bicarbonate pools, there is a delay in researchers ability to utilize the 13 CO 2 being collected at the mouth (144) and the baseline shift in 13 CO 2 production from endogenous sources (79). It has also been suggested that the use of 13 C may lead to overestimations of CHO oxidation while the use of 14 C may result in underestimations of CHO oxidation (106). In some instances the estimates of exogenous carbohydrate oxidation may exceed the amount of carbohydrate ingested. Peronnet et al (1990) described this error as an over simplification of the assumption that all of the excess 13 CO 2 is the result of the ingestion of the exogenously labeled substrate (106). The North American diet consists of many food ingredients, such as maze and cane sugar, which naturally contain high levels of 13 C. This can result in increased amounts of 13 C being stored in endogenous carbohydrate stores. As the endogenous stores of carbohydrate are oxidized for energy, the 13 C in it is expired as additional 13 CO 2 . This has been reported by Wolfe (1984) (141) and repeatedly by Massicotte (78; 80; 81). Overestimations of exogenous carbohydrate are less likely when the exogenous carbohydrate is highly enriched (106). Researchers have restricted participants? food selections and artificially enriched beverages with 15 labeled 13 C-glucose and 13 C-fructose to overcome the naturally occurring elevations in 13 C (50; 79-81; 107; 108). Since products containing naturally high levels of 13 C are not as widely used in Europe, many 13 C carbohydrate oxidation studies conducted in Europe have utilized corn-derived glucose, crystalline fructose, and/or sugar cane-derived sucrose to analyze exogenous carbohydrate oxidation successfully (59; 60; 63; 64; 67- 69). Not all positive physiological changes resulting from carbohydrate ingestion translate to improved performance. Mitchell (1989) found that ingestion of a beverage delivering 111 g?hr -1 of carbohydrate elevated blood glucose more than other treatments but this treatment?s performance measure was no better than placebo (89). Reviewing 73 studies with 110 carbohydrate treatments, carbohydrate improved performance compared to a non-carbohydrate treatment in 61 of the 110 treatments (1; 3; 4; 24; 37; 39; 45; 66; 77; 88; 93; 98; 99; 101; 116; 127; 133; 134; 139; 143). Unfortunately, variations in carbohydrate type, concentration, delivery form, delivery schedule, and testing protocol used in many of the studies make comparisons of dosage benefits difficult. A number of studies have directly or indirectly addressed the question of whether a dose-response relationship exists between carbohydrate intake and performance (84; 89; 90; 92; 95). A dose-response relationship has yet to be found (92; 93). Carbohydrate?s impact on exercise performance has been assessed using various methods. The most common method to analyze the impact of carbohydrate on exercise performance is cycling exercise. Reasons cycling is the most common endurance exercise mode to study carbohydrate feeding may result from difficulty of ingesting fluid 16 and increased incidence of gastric discomfort when running (19). The most common measures used to assess exercise performance are rides to exhaustion (12; 17; 20; 21; 29; 31; 34; 42; 49; 56; 57; 84; 99; 121; 132; 142; 143) amount of work completed in a set time (36; 37; 43; 89; 90; 97),and time to complete a set amount of work (3; 13; 33; 39; 87; 88; 92; 93; 95; 98; 116). Studies using time to exhaustion to analyze the impact of nutrient ingestion on exercise performance have found many performance enhancements when ingesting carbohydrate as compared to a placebo (12; 17; 20; 21; 29; 31; 34; 42; 49; 56; 57; 84; 99; 121; 132; 142; 143). Coyle et al (1986) and Nicholas et al (1995) reported carbohydrate ingestion increased time to fatigue by 33% (29; 99). In 1984, Hargreaves et al reported a 45% increase in time to exhaustion when carbohydrate was ingested during exercise (49). Similarly, using the amount of work has repeatedly demonstrated a performance improvement when carbohydrate is ingested (36; 37; 43; 89; 90; 97). While time to exhaustion and amount of work completed in a set amount of time seem to be good methods to demonstrate relationships in nutrient ingestion and performance, they both lack related exercise situations. Based on the findings of this previous research, this study examined the physiological and exercise performance responses of cyclists during a 2-hour constant load ride followed by a simulated 20-km time trial to determine the impact of carbohydrate delivery rates below what is typically recommended (60 g?hr -1 ) for prolonged exercise. Participants completed four trials including a trial with no carbohydrate and three trials where carbohydrate was ingested in a drink at a rate of 15 g?h -1 , 30 g?h -1 , and 60 g?h -1 . 17 III: METHODS Participants: Twelve trained male cyclists and triathletes participated in this study. Mean and standard error of age, height, mass, and peak oxygen uptake (V & O 2 PEAK ) were 31.7 ? 1.1 yrs, 1.82 ? 0.02 m, 77.6 ? 2.0 kg, and 4.12 ? 0.09 l?min -1 , respectively. Participants served as their own controls for the study. All participants read and signed an informed consent approved by the Human Subject Review Committee prior to beginning the study. Preliminary Testing: Peak oxygen uptake (V & O 2 PEAK ) was determined using an increasing resistance, multistage cycling test with 30 second Douglas Bags collected during the last 30 seconds of each stage. Expiratory gases were analyzed using an Ametek S-3A/I Oxygen and Ametek CD-3A Carbon Dioxide Analyzers (Naperville, IL). Expired volume was measured with a spirometer (Vacumed Inc., Ventura CA). A regression analysis of the V & O 2 -workload relationship determined the exercise workloads for lactate threshold testing. The participants? two hour ride workload was set at 95% of the workload that would elicit a 4 mmol?L -1 blood lactate. Preliminary research in our lab found that this was the highest intensity most participants could maintain for two hours of cycling. Subjects exercised for 3-minute stages at 55, 60, 65, 70, 75, 80, 85, and 90% of their 18 V & O 2PEAK with blood samples taken for lactate analysis at the end of each stage. Blood was analyzed using a whole blood analyzer (Gem Premier 3000, Instrumentation Laboratory, Lexington, MA). All testing was performed with the participants exercising on their own bicycle affixed to a CompuTrainer? Pro (RacerMate Inc, Seattle WA). Participant Orientation: Participants were required to perform three familiarization course rides prior to beginning the actual trials, with at least seven days between course rides. The first familiarization was performed to allow the participant to become familiarized with the 20-km time trial course. The second familiarization was performed to allow the participant to familiarize himself with the feel of a 2-hour ride followed by the 20-km time trial. This model is commonly used in metabolic fuel investigations but also has application to competitive cycling events. The third and final familiarization was performed to allow the participant to experience the testing procedures. Experimental Protocol: Participants completed four exercise trials with randomized interventions separated by at least seven days. Trials were conducted in a laboratory maintained at 20- 25?C, 35-40% relative humidity. Participants were instructed to record and maintain the same diet and abstain from exercise 24 hours before each trial. Participants reported to the lab after a 10-hour overnight fast. After arriving at the lab, participants voided and body weights were recorded. A registered nurse then inserted an intravenous catheter (BD Insyte? Autoguard?, 19 Becton, Dickinson Infusion Therapy Systems Inc., Sandy UT) into an antecubital vein with a three-way stopcock (Solution-Plus?, Mansfield, MA) to collect baseline blood samples and allow for blood sampling throughout the trial. Participants performed a standardized ten-minute ride at 100 watts to warm up and prepare the trainers for calibration. Following the 10-minute ride the CompuTrainers? were calibrated according to the manufacturer?s recommendations. On average, participants workload was set at 227.5 ? 2.2 watts, after which they began a two hour constant load ride on the CompuTrainer? with V & O 2, ratings of perceived exertion, and heart rate collected every 15 minutes and blood samples collected every 15 minutes during the final hour (Figure 1). After completing the 2-hour ride participants stopped pedaling and were allowed off the bike for one and a half minutes. Two-minutes after the cessation of the two hour constant load ride, participants began the simulated 20-km time trial. For the time trial participants were instructed to complete the 20-km course (Figure 2) as quickly as possible. The undulating course was designed using the CompuTrainer? 3D computer program. Participants were aware of the approximate distance traveled and remaining from a course profile showing their position on the course but no verbal stimuli or other information was given. Resistance varied throughout the ride based on the inclination of the course and the participants? speed and gearing, similar to that experienced when cycling outdoors. Participants were allowed to change gears at will throughout the course of the ride. 20 Intervention Beverages: Participants completed four trials during this study. The intervention beverages tested during the trials were 1) 250 mL of Placebo with electrolytes (PLA); 2) 250 ml of 1.5% glucose with electrolytes (15 g?hr -1 ); 3) 250 ml of 3.0% glucose with electrolytes (30 g?hr -1 ); and 4) 250 ml of 6.0% glucose with electrolytes (60 g?hr -1 ). Beverages were consumed every 15 minutes during the 2-hour ride. All beverages were formulated to be identical in flavor and sweetness, and kept at a temperature of 1?C to minimize differences in taste. The test beverages were given to the participant in opaque plastic containers. To allow for the calculation of exogenous glucose oxidation, uniformly labeled 13 C-glucose (Isotec?, Miamisburg, OH) was added at 1.8 mg?g -1 of glucose contained within the experimental beverage. This resulted in beverages with high abundances of 13 C (15: 121 Pee Dee Bellemnitella (? [?- 13 C]PDB-1), 30: 136 ? [?- 13 C]PDB-1, and 60: 151 ? [?- 13 C]PDB-1). Data Collection: Heart Rate and Perception Heart rate was measured with a chest strap and watch telemetry system (Polar Electro Inc., Lake Success, NY). Heart rate and a Borg Scale was used to determine athletes? perceived exertion was recorded every 15 minutes during the two hour ride. 21 Physiological Measures and Metabolites A 60-second expired air sample was collected every 15 minutes during the 2-hour ride. The expired air sample was analyzed for oxygen and carbon dioxide partial pressures and the expired volume was measured using a flow meter. With the expired air measures, V & O 2 , V & CO 2 , and RER were calculated, neglecting the small contribution of protein oxidation to the energy yield (71; 107). Total carbohydrate (TCO) and fat oxidation rates were calculated using equation 1 and 2, respectively: (28) (g?min -1 ) = (4.59 ? V & CO 2 ) ? (3.23 ? V & O 2 ) (Equation 1) (g?min -1 ) = 1.70 ? (V & O 2 ? V & CO 2 ) (Equation 2) Whole blood samples were used to measure plasma lactate, hematocrit, and hemoglobin using a Gem Premier 3000 whole blood analyzer from Instrumentation Laboratory. Plasma glucose was measured using a liquid glucose (Hexokinase) Reagent Set kit (Pointe Scientific, Canton, MI). Insulin was measured using a Human Insulin ELISA kit (Millipore Corp, St. Charles, MO). Free-fatty acids were measured using a non-essential fatty acid HR2 series reagents kit (Wako Diagnostics, Richmond, VA). Cortisol was measured using a Cortisol kit (Pointe Scientific, Canton, MI). All samples were analyzed on a Multisample Spectrophotometer (Synergy HT, Bio-tek Instruments, Winooski, MA). 22 Tracer To determine exogenous carbohydrate oxidation rate expired gas was captured in a 10 mL vacutainer (BD Vacutainer?, Franklin Lakes, NJ) for analysis of expired 13 CO 2 / 12 CO 2 . Expired 13 CO 2 / 12 CO 2 ratio was measured using a BreathMat Plus (Finnigan MAT). Exogenous glucose oxidation rate (EGO) was calculated using equation 3: (70; 108) (g?min -1 ) = V & CO 2 ? R OBS ? R REF ? 1 (Equation 3) R ING ? R REF k Where R ? isotopic ratio, R OBS ? The observed 13 C/C ratio in the breath, R ING ? The 13 C/C ratio of the beverage ingested, R REF ? The baseline breath 13 C/C ratio, and k (0.747 L?g -1 ) is the amount of V & CO2 provide by the oxidation of 1 g of glucose. During the second hour of exercise, plasma samples were collected to allow for the calculation of exogenous glucose present in the blood, plasma glucose oxidation, liver glucose oxidation, and muscle glucose oxidation. These calculations were made only during the final 60 min of the 2-hour constant load ride allowing 60 min of equilibration. This 60 min equilibration is based on observations that 13 C/ 12 C in expired CO 2 equilibrates slowly with the 13 C/ 12 C in the CO 2 produced in tissues (103), that 13 C provided from 13 C-glucose is not irreversibly lost in tricarboxylic acid cycle intermediates (118) and/or bicarbonate (128) pools, and that the 13 CO 2 recovery in expired gases is complete or almost complete. 23 Plasma was deproteinized with barium hydroxide (0.3 N) and zinc sulfate (0.3 N). Glucose was then separated from the plasma by double-bed ion-exchange chromatography (AG 50W-X8 H + and AG 1-X8 chloride, 200-400 mesh; Bio-Rad, Mississauga, ON, Canada). The elute was then evaporated to dryness (Virtis Research Equipment, New York, NY) and combusted with copper oxide for 60 minutes at 400?C. The CO 2 was recovered from the glucose combustion for isotopic analysis. The measurement of 13 C/ 12 C in the CO 2 coming from combusted glucose was then performed using mass spectrometry (Prism, Manchester, UK). The isotopic ratio of the combusted plasma glucose was expressed in percentage difference (Equation 4) by comparison with the PDB Chicago Standard: ? [?- 13 C]PDB-1 = [Rspl/Rstd) ? 1] X 1,000 (Equation 4) Where Rspl ? 13 C/C ratio in the sample and Rstd ? 13 C/C ratio in the standard (1.1237 ?), respectively (16). The percentage of plasma glucose derived from exogenous glucose was calculated using equation 5: (%) = R GLU ? R REF ? 100 (Equation 5) R ING ? R REF Where R ? isotopic ratio, R GLU ? The observed 13 C/C ratio in the blood, R ING ? The 13 C/C ratio of the beverage ingested, and R REF ? The baseline blood 13 C/C ratio. 24 Plasma glucose oxidation (PGO) was calculated using equation 6: (108) (g?min -1 ) = ____Plasma Glucose being Oxidized_____ (Equation 6) Exogenous Plasma Glucose Concentration Liver glucose oxidation (LGO) was calculated using equation 7: (28; 50) (g?min -1 ) = PGO ? EGO (Equation 7) Muscle glycogen oxidation (expressed in grams of glucose/min) was calculated using equation 8: (28; 50) (g?min -1 ) = TCO - PGO (Equation 8) Statistical Analysis: All data are expressed as mean ? standard error. Statistical analysis was performed using SPSS version 13 (SPSS Inc. Chicago, IL). Data were analyzed with a univariate ANOVA. When ANOVA reported significant interactions, a Duncan post-hoc analysis was performed. Significance was set at p ? 0.05. 25 IV: RESULTS Physiological Measures: Heart rate increased significantly over the duration of the 2-hour constant load ride (Figure 3). RPE significantly increased over the duration of the 2-hour constant load ride (Figure 4). RPE was significantly higher during 15 g?hr -1 as compared to all other trials and 30 g?hr -1 was significantly lower than PLA. V & O 2 during the 2-hour constant load ride averaged 3.17 ? 0.08, 3.21 ? 0.09, 3.19 ? 0.10, and 3.16 ? 0.09 for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 , respectively (Figure 5). This average V & O 2 of the four trials represented 77.2 ? 0.9% of the participants? V & O 2 PEAK . There was a significant reduction in RER (Figure 6) and carbohydrate oxidation (Figure 7) while fat oxidation significantly increased (Figure 8) during the 2-hour constant load ride (p ? 0.05). RER and carbohydrate oxidation was significantly higher while fat oxidation were significantly lower in the 60 g?hr -1 trial as compared to all other trials (p ? 0.05). Blood, Hormone, and Metabolite Measures: There was no difference in baseline blood glucose, insulin, or free-fatty acid measures. There were significant reductions in plasma glucose (Figure 9) and insulin (Figure 10) levels during the last hour of the 2-hour constant load ride. With reductions in carbohydrate ingestion below 30 g?hr -1 , plasma glucose levels declined at a significantly greater rate as carbohydrate ingestion declined. Plasma glucose levels were 26 significantly lower in the PLA trial as compared to the 15 g?hr -1 trial. Plasma insulin levels were significantly higher in the 60 g?hr -1 trial as compared to all other trials. There was a significant rise in plasma free-fatty acid levels during the last hour of the 2-hour constant load ride (Figure 11). There was no difference in plasma free-fatty acid levels when comparing the PLA and 15 g?hr -1 trials. As carbohydrate ingestion increased above 15 g?hr -1 , plasma free-fatty acid levels significantly decreased as glucose ingestion increased. There was a significant increase in plasma lactate levels during the 2-hour constant load ride (Figure 12), but no difference during exercise and no difference between treatments. Average lactate levels during exercise were 2.96 ? 0.47 mmol?L -1 , 2.99 ? 0.55 mmol?L -1 , 2.75 ? 0.53 mmol?L -1 , and 2.83 ? 0.52 mmol?L -1 for PLA, 15 g?hr - 1 , 30 g?hr -1 , and 60 g?hr -1 , respectively. Plasma cortisol levels rose significantly during the second hour of the 2-hour constant load ride (Figure 13). There was no difference in plasma cortisol levels. Average cortisol levels during the 2-hour constant load ride were 243.05 ? 41.24, 249.33 ? 39.15, 236.40 ? 37.95, and 242.28 ? 35.09 for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 , respectively. Exogenous and Endogenous Glucose Utilization: The expired 13 C/C ratio significantly increased during the 2-hour constant load ride with all treatments being significantly different at the beginning of the second hour of exercise (Figure 14). The percentage of exogenous glucose in the plasma increased significantly as exogenous carbohydrate intake increased (Figure 15). During the 2-hour constant load ride, exogenous glucose oxidation significantly increased as glucose ingestion rate increased. Exogenous glucose oxidation rates during the last 30 minutes of 27 the constant load cycling were 0.26 ? 0.05, 0.44 ? 0.04 and 0.66 ? 0.07 g?min -1 for 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 ingestion rates, each being significantly different from all others (p ? 0.001). There was no difference in total plasma glucose oxidation in the 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 trials. Since 13 C was not given in the PLA trial we are unable to compare plasma glucose oxidation in the PLA trial. There was, however, a significant increase in liver glycogen oxidation during the last hour of the 2-hour constant load ride. Liver glycogen oxidation rate was highest when consuming 15 g?hr -1 (0.63 ? 0.13 g?min -1 ) followed by 30 g?hr -1 (0.51 ? 0.12 g?min -1 ) and 60 g?hr -1 (0.42 ? 0.08 g?min -1 ), all significantly different from one another at a p ? 0.05 level. There was also significant reduction in muscle glycogen oxidation during the last hour of the 2-hour constant load ride with no significant differences found between the 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 trials. No tracer was given in the PLA to examine muscle glycogen oxidation during the PLA trial. (Figures 16) Performance Measures: The time required to complete the 20-km time trial was 36.39 ? 0.84 min, 35.23 ? 0.80 min, 34.99 ? 0.75 min, and 34.69 ? 0.62 min for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 , respectively. The change in time required to complete the 20-km time trial compared to PLA are shown in Figure 17. The average wattage during the time trial for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 was 212.0 ? 9.5 W, 227.9 ? 10.7 W, 229.7 ? 10.6 W, and 234.2 ? 9.4 W, respectively. The change in average wattage during the 20-km time trial compared to PLA are shown in Figure 18. PLA required significantly more time to complete the time trial and resulted in a significantly lower average wattage 28 during the time trial compared to 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 (p ? 0.05). However, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 were not significantly different from one another. 29 Figure 1: Order of test measures Diagram of the testing protocol. 0 15 30 45 60 75 90 105 120 RPE RPE RPE RPE RPE RPE RPE RPE HR HR HR HR HR HR HR HR V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 Blood Blood Blood Blood Blood Blood Drink Drink Drink Drink Drink Drink Drink Drink Constant Load Ride 20-K TT 30 Figure 2: 20-kilometer course profile Course profile of the 20-km time trial. 31 Figure 3: Heart rate during 2 hour constant load ride 140 145 150 155 160 0 153045607590105120 Minutes BPM PLA 15 g/min 30 g/min 60 g/min Heart rate response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). There were no significant differences seen between treatments. There is a significant main effect for time (p < 0.05). 32 Figure 4: Ratings of perceived exertion during the 2-hour constant load ride 6 8 10 12 14 16 18 20 0 153045607590105120 Minutes RPE PLA 15 g/min 30 g/min 60 g/min Ratings of perceived exertion during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). PLA is significantly different than 15 g?hr -1 and 30 g?hr -1 , 15 g?hr -1 is significantly different than PLA, 30 g?hr -1 , and 60 g?hr -1 , There is a significant main effect for time (p < 0.05). 33 Figure 5 ? V & O 2 during exercise 2.50 2.75 3.00 3.25 3.50 15 30 45 60 75 90 105 120 Minutes L?min -1 PLA 15 g/min 30 g/min 60 g/min Oxygen uptake response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA and 60 g?hr -1 . b significantly higher than 60 g?hr -1 . There is a significant main effect for time (p < 0.05). a b 34 Figure 6 ? Respiratory exchange ratio during exercise 0.85 0.90 0.95 1.00 15 30 45 60 75 90 105 120 Minutes RER PLA 15 g/min 30 g/min 60 g/min Respiratory exchange ratio response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA, 15 g?hr -1 and 30 g?hr -1 . There is a significant main effect for time (p < 0.05). a 35 Figure 7 ? Carbohydrate oxidation during exercise 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 15 30 45 60 75 90 105 120 Minutes g?min -1 PLA 15 g/min 30 g/min 60 g/min Carbohydrate oxidation response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA, 15 g?hr -1 ,and 30 g?hr -1 . There is a significant main effect for time (p < 0.05). a 36 Figure 8 ? Fat oxidation during exercise 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 15 30 45 60 75 90 105 120 Minutes g/min PLA 15 g/hr 30 g/hr 60 g/hr Fat oxidatoion during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly lower than PLA, 15 g?hr -1 ,and 30 g?hr -1 . There is a significant main effect for time (p < 0.05). a 37 Figure 9 ? Blood glucose response during exercise 60 70 80 90 100 110 120 60 75 90 105 120 Minutes mg?dL -1 PLA 15 g/min 30 g/min 60 g/min Plasma glucose response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than 0; b significantly higher 15. There is a significant main effect for time (p < 0.05). ab ab a 38 Figure 10 ? Insulin response during exercise 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 60 75 90 105 120 Minutes mU?L -1 PLA 15 g/min 30 g/min 60 g/min Plasma insulin response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than all other treatments (p < 0.05). There is a significant main effect for time (p < 0.05). a 39 Figure 11 ? Free fatty acid response during exercise 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 60 75 90 105 120 Minutes mE q?L -1 PLA 15 g/min 30 g/min 60 g/min Serum free-fatty acid response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly lower than 0 and 15; b significantly lower 30. (p < 0.05). There is a significant main effect for time (p < 0.05). a ab 40 Figure 12 ? Lactate during 2-hour constant load exercise 1.0 1.5 2.0 2.5 3.0 3.5 4.0 60 75 90 105 120 Minutes mmo l ?L -1 PLA 15 g/min 30 g/min 60 g/min Plasma lactate response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). There were no significant differences seen between treatments. There is a significant main effect for time (p < 0.05). 41 Figure 13 ? Cortisol response during exercise 150 200 250 300 350 60 75 90 105 120 Minutes IU?L -1 PLA 15 g/min 30 g/min 60 g/min Plasma cortisol response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). There were no significant differences seen between treatments. There is a significant main effect for time (p < 0.05). 42 Figure 14 ? 13 C/C ratio in expired air during exercise -30 -25 -20 -15 -10 -5 0 5 10 15 0 153045607590105120 Minutes ? [ ? -13C]PDB -1 PLA 15 g/hr 30 g/hr 60 g/hr Expired PDB in the breath during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a 15 g?min -1 significantly lower than PLA, 30 g?min -1 and 60 g?min -1 , b PLA significantly lower than 15 g?min -1 , 30 g?min -1 , and 60 g?min -1 , c 15 g?min -1 significantly lower than 30 g?min -1 and 60 g?min -1 , and d 30 g?min -1 significantly lower than 60 g?min -1 . a bc bcd bcd bcd bcd bcd 43 Figure 15 ? Percent plasma glucose coming from an exogenous source 0 10 20 30 40 50 60 70 80 90 100 PLA 15 g/min 30 g/min 60 g/min Trial % 60 min 90 min 120 min Percent plasma glucose coming from an exogenous source during the last hour of a 2- hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). All trials are significantly different from each other. There were no significant time differences within trials. 44 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 Time 60 90 120 60 90 1 20 60 90 120 minute s Tr e a t men t 15 g ? h -1 30 g ? h -1 60 g ? h -1 Musc le g l y c o gen L i ve r Gluc os e Ex og e nous Gluc ose 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 g? m i n -1 F i g u r e 16 ? S ou r c e of ox idiz e d c a r boh y d ra t e duri n g th e l a st hour of e x e r c i se whe n c onsu m in g g l u c o s e a t 15, 30 , a nd 60 g ? h -1 . Car boh y d rat e utiliz a tion during the fin a l hour of a 2-hour ride a t 9 5 % of th e worklo a d th a t e l i c its a 4 mmol bloo d lac t a t e r e s p o nse whe n c onsu m in g 15, 3 0, a nd 60 g of g l u c ose an h our. Va lu e s a r e m e a n s ( S E) . a sig n if ic antl y l e ss th an 15 g ? h -1 ; b s i g n i f ic a n tly le ss tha n 30 g ? h -1 ; c sig n i f ic antl y gre a t e r tha n 15 g?h -1 ; d si g n ifica n tl y g r ea t e r t h a n 30 g ? h -1 . (p < 0.0 5) a c ab cd 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 Time 60 90 120 60 90 1 20 60 90 120 minute s Tr e a t men t 15 g ? h -1 30 g ? h -1 60 g ? h -1 Musc le g l y c o gen L i ve r Gluc os e Ex og e nous Gluc ose 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 g? m i n -1 F i g u r e 16 ? S ou r c e of ox idiz e d c a r boh y d ra t e duri n g th e l a st hour of e x e r c i se whe n c onsu m in g g l u c o s e a t 15, 30 , a nd 60 g ? h -1 . Car boh y d rat e utiliz a tion during the fin a l hour of a 2-hour ride a t 9 5 % of th e worklo a d th a t e l i c its a 4 mmol bloo d lac t a t e r e s p o nse whe n c onsu m in g 15, 3 0, a nd 60 g of g l u c ose an h our. Va lu e s a r e m e a n s ( S E) . a sig n if ic antl y l e ss th an 15 g ? h -1 ; b s i g n i f ic a n tly le ss tha n 30 g ? h -1 ; c sig n i f ic antl y gre a t e r tha n 15 g?h -1 ; d si g n ifica n tl y g r ea t e r t h a n 30 g ? h -1 . (p < 0.0 5) a c ab cd 45 Figure 17: Change in 20-km time trial completion time in relation to PLA -7 -6 -5 -4 -3 -2 -1 0 1 2 3 15 30 60 Treatment Minutes Box plots of change in duration in the 20-km time trial performance following a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response when consuming glucose at various rates. The top and bottom of the box represents the 75 th and 25 th percentile. The whiskers capture the range of performance times for the entire group of subjects. The black line in the box corresponds to the median performance value. No differences were observed between glucose treatments. No differences were observed between glucose treatments. All glucose treatments were significantly faster than placebo. 46 Figure 18: Change in 20-km time trial average wattage in relation to PLA -40 -20 0 20 40 60 80 100 15 30 60 Treatment Watts Box plots of change in average wattage in the 20-km time trial workrate following a 2- hour ride at 95% of the workload that elicits a 4 mmol blood lactate response when consuming glucose at various rates. The top and bottom of the box represents the 75 th and 25 th percentile. The whiskers capture the range of performance times for the entire group of subjects. The black line in the box corresponds to the median performance value. No differences were observed between glucose treatments. No differences were observed between glucose treatments. All were significantly faster than placebo. 47 V: IMPACT OF LOW DOSE GLUCOSE INGESTION ON EXOGENOUS GLUCOSE OXIDATION, SUBSTRATE UTILIZATION, AND EXERCISE ABSTRACT This study investigated the impact of glucose ingestion at rates of 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 on carbohydrate metabolism and exercise performance. Twelve cyclist/triathletes cycled at ~75% V & O 2 PEAK for two hours while ingesting glucose drinks delivering 15, 30, and 60 g?hr -1 or a placebo. Glucose drinks were extrinsically labeled with 1.8 mg?g -1 U- 13 C-glucose. Expired breath samples and blood samples were collected every 15 minutes for future analyses. Immediately following the two hour constant load exercise session, participants completed a 20-km time-trial as quickly as possible. Exogenous glucose oxidation rose significantly as ingestion rate increased. Blood glucose and insulin were highest when ingesting 60 g?hr -1 while free fatty acids were the lowest. Insulin and free fatty acid responses for placebo and the 15 g?hr -1 trial were virtually identical. Liver glucose oxidation was significantly reduced by increasing glucose ingestion. Relative to placebo, glucose ingestion improved (p < 0.01) time-trial performance with no statistical difference between glucose doses. The findings of this study indicate that increasing glucose ingestion rate increases oxidation of exogenous glucose and spares liver-derived glucose. This study also demonstrates that 60 g?hr -1 provided the most consistent improvements in performance, even though performance 48 can be improved when ingesting only 15 g?hr -1 during exercise lasting approximately 150 minutes. Key Words: cycling, exogenous carbohydrate oxidation, substrate utilization, performance INTRODUCTION Fatigue during prolonged exercise has been associated with hypoglycemia and depletion of muscle glycogen (6; 11; 19; 49). There is a great deal of evidence demonstrating the ergogenic effect of consuming carbohydrate before and during endurance exercise (6; 8; 11; 12; 16; 24; 51; 57; 58). The American College of Sports Medicine (ACSM) and the National Athletic Trainers Association (NATA) both have fluid replacement position stands that promote the benefit of carbohydrate intake during exercise. Both position stands suggest carbohydrate ingestion rates of 30-60 g?hr -1 (5; 46). These position statements and experts? findings have suggested the need for carbohydrates during prolonged exercise, but the specific carbohydrate ingestion rate for optimal performance benefits have yet to be defined. Many researchers using various techniques have investigated the question to identify the carbohydrate ingestion rate that optimizes exercise performance and substrate utilization (6; 11; 12; 21; 26). Results indicate that time to exhaustion is prolonged when carbohydrate is consumed during exercise (6; 11; 12; 16; 51; 57; 58). Carbohydrate intake during exercise has also been shown to reduce time required to complete a set distance or work volume (1; 2; 35; 36). 49 Many of the improvements in performance experienced with carbohydrate ingestion have been associated to the maintenance of carbohydrate oxidation late during exercise (11; 53; 57). Carbohydrate oxidation is maintained through the oxidation of exogenous carbohydrate sources (30; 37; 43), the maintenance of blood glucose (1; 11; 53), and the sparing of endogenous carbohydrate (27; 37; 43). Most research suggests that liver glucose and not muscle glycogen is the endogenous carbohydrate source spared through exogenous carbohydrate ingestion. Liver glucose has been repeatedly shown to be spared through the ingestion of carbohydrate prior to and during exercise (3; 26; 30). While some running studies and a few cycling studies have demonstrated a sparing of muscle glycogen (37; 50; 51), most cycling studies do not report the sparing of muscle glycogen when carbohydrate is ingested during exercise (6; 11; 12; 16; 32). Improvements in exercise performance have been reported with carbohydrate intakes ranging from 18 to 180 g?h -1 during exercise (12; 16; 32; 33; 36; 58). Many studies have tested carbohydrate ingestion rates above those recommended by the ACSM and NATA position stands in hopes of optimizing exercise performance and exploring the possibility of a carbohydrate/performance dose response. These studies have failed to demonstrate greater enhancements in performance with carbohydrate ingestion rates above ACSM and NATA recommended ingestion rates compared to the recommended rate range (14; 32; 35). While the impact of increasing carbohydrate ingestion rate has not been demonstrated, less is known about metabolic changes occurring at lower carbohydrate ingestion rates, the minimum level of carbohydrate intake needed for improved exercise performance, or if there is a dose response to varying carbohydrate 50 levels within and below the ingestion rates recommended by the ACSM and NATA position stands (16; 35; 55; 58). While it might be logical that providing increased levels of carbohydrate will provide additional energy for exercise, carbohydrate ingestion rates above those defined in the ACSM and NATA position stands have also been associated with an increased incidence of stomach discomfort and stomach upset which may result in diminished exercise performance (42; 45; 52). Scientists have demonstrated increasing carbohydrate ingestion rate above 1.2 g?min -1 , with a single carbohydrate form, does not further increase exogenous carbohydrate oxidation (21; 53). Research has also demonstrated that when only glucose is ingested the body is able to utilize that glucose at a rate of about 1 g?min -1 (23; 26; 27; 53). When multiple forms of carbohydrates (i.e. glucose or glucose polymers, and fructose) are consumed, glucose oxidation rates have been reported to be up to and above 1.5 g?min -1 (21; 23; 26; 27; 53). This study examined the physiological and exercise performance responses of cyclists during a 2-hour constant load ride followed by a simulated 20-km time trial to determine the impact of carbohydrate delivery rates below what is typically recommended (60 g?h -1 ) for prolonged exercise. Participants completed four trials including a trial with no carbohydrate and three trials where carbohydrate was ingested in a drink at a rate of 15 g?h -1 , 30 g?h -1 , and 60 g?h -1 . 51 METHODS Participants: Twelve trained male cyclists and triathletes participated in this study. Mean and standard deviation age, height, mass, and peak oxygen uptake (V & O 2 PEAK ), were 31.7 ? 1.1 yrs, 1.82 ? 0.02 m, 77.6 ? 2.0 kg, and 4.12 ? 0.09 l?min -1 , respectively. Participants served as their own controls for the study. All participants read and signed an informed consent approved by the Human Subject Review Committee prior to beginning the study. Preliminary Testing: V & O 2 PEAK was determined using an increasing resistance, multistage cycling test with 30 second Douglass Bags collected during the last 30 seconds of each stage. Expiratory gases were analyzed using Ametek S-3A/I Oxygen and Ametek CD-3A Carbon Dioxide Analyzers (Naperville, IL) and expired volume was measured with a spirometer (Vacumed Inc., Ventura CA). A regression analysis of the V & O 2 -workload relationship determined the exercise workloads for lactate threshold testing. The participants? two hour ride workload was set at 95% of the workload that would elicit a 4 mmol?L -1 blood lactate. Preliminary research in our lab found that this was the highest intensity most participants could maintain for two hours of cycling. Subjects exercised for 3-minute stages at 55, 60, 65, 70, 75, 80, 85, and 90% of their V & O 2 PEAK with blood samples taken for lactate analysis at the end of each stage. Blood was analyzed using a whole blood analyzer (Gem Premier 3000, Instrumentation Laboratory, Lexington, MA). All testing was performed with the participants exercising on their own bicycle affixed to a CompuTrainer? Pro (RacerMate Inc, Seattle WA). 52 Participant Orientation: Participants were required to perform three familiarization course rides prior to beginning the actual trials, with at least seven days between course rides. The first familiarization was performed to allow the participant to become familiarized with the 20-km time trial course. The second familiarization was performed to allow the participant to familiarize himself with the feel of a 2-hour ride followed by the 20-km time trial. This model is commonly used in metabolic fuel investigations but also has application to competitive cycling events. The third and final familiarization was performed to allow the participant to experience the testing procedures. Experimental Protocol: Participants completed four exercise trials with randomized interventions separated by at least seven days. Participants were instructed to record and maintain the same diet and abstain from exercise 24 hours before each trial. Participants reported to the lab after a 10-hour overnight fast. After arriving at the lab, participants voided and body weight were recorded. A registered nurse then inserted an intravenous catheter (BD Insyte? Autoguard?, Becton, Dickinson Infusion Therapy Systems Inc., Sandy UT) into an antecubital vein with a three-way stopcock (Solution-Plus?, Mansfield, MA) to collect baseline blood samples and allow for blood sampling throughout the trial. Environmental conditions were maintained at 20-25?C, 35-40% relative humidity throughout the experiment. Participants performed a standardized ten-minute ride at 100 watts to warm-up and prepare the CompuTrainers? for calibration. Following the 10- minute ride the CompuTrainers? were calibrated according to the manufacturer?s 53 recommendations. On average, participants 2-hour workload was set at 227.5 ? 2.2 watts after which they began a two hour constant load ride on the CompuTrainer? with V & O 2 and heart rate collected every 15 minutes and blood samples collected every 15 minutes during the final hour (Figure 1). After completing the 2-hour ride participants stopped pedaling and were allowed off the bike for one and a half minutes. Two-minutes after the cessation of the two hour constant load ride, participants began the simulated 20-km time trial. For the time trial participants were instructed to complete the 20-km course (Figure 2) as quickly as possible. This undulating course was designed using the CompuTrainer? 3D computer program. Participants were aware of their position on the course but no verbal stimuli or other information was given by the investigators. Resistance varied throughout the ride based on the inclination of the course and the participants? speed and gearing, similar to that experienced when cycling outdoors. Participants were allowed to change gears at will throughout the course of the ride. Intervention Beverages: Participants completed the 2-hour exercise sessions consuming one of the four treatments over four trials during this study. The beverage treatments tested during the trials were 1) 250 mL of placebo with electrolytes (PLA); 2) 250 ml of 1.5% glucose with electrolytes (15 g?hr -1 ); 3) 250 ml of 3.0% glucose with electrolytes (30 g?hr -1 ); and 4) 250 ml of 6.0% glucose with electrolytes (60 g?hr -1 ) consumed every 15 minutes of the 2- hour ride. All beverages were formulated to be identical in flavor, sweetness and kept at a temperature of 1?C to minimize differences in taste. The test beverages were given to 54 the participant in opaque plastic containers. To allow for the calculation of exogenous glucose oxidation, uniformly labeled 13 C-glucose (Isotec?, Miamisburg, OH) was added at 1.8 mg?g -1 of glucose contained within the experimental beverage. This resulted in beverages with high abundances of 13 C (15: 121 Pee Dee Bellemnitella (? [?- 13 C]PDB- 1), 30: 136 ? [?- 13 C]PDB-1, and 60: 151 ? [?- 13 C]PDB-1). Data Collection: Physiological Measures and Metabolites Heart rate was measured every 15 minutes during the two hour ride with a chest strap and watch telemetry system (Polar Electro Inc., Lake Success, NY). A 60-second expired air sample was collected every 15 minutes during the 2-hour ride. The expired air sample was analyzed for oxygen and carbon dioxide fraction and the expired volume was measured using a flow meter. With the expired air measures, V & O 2 , V & CO 2 , and RER were calculated. Total carbohydrate (TCO) and fat oxidation rates were calculated, neglecting the small contribution of protein oxidation to the energy yield (75; 116), using equation 1 and 2, respectively: (10) (g?min -1 ) = (4.59 ? V & CO 2 ) ? (3.23 ? V & O 2 ) (Equation 1) (g?min -1 ) = 1.70 ? (V & O 2 ? V & CO 2 ) (Equation 2) Plasma glucose was measured using a liquid glucose (Hexokinase) Reagent Set kit (Pointe Scientific, Canton, MI). Insulin was measured using a Human Insulin ELISA kit (Millipore Corp, St. Charles, MO). Free-fatty acids were measured using a non- 55 essential fatty acid HR2 series reagents kit (Wako Diagnostics, Richmond, VA). All samples were analyzed on a Multisample Spectrophotometer (Synergy HT, Bio-tek Instruments, Winooski, MA). Tracer To determine exogenous carbohydrate oxidation rate expired gas was captured in a 10 mL vacutainer (BD Vacutainer?, Franklin Lakes, NJ) for analysis of expired 13 CO 2 / 12 CO 2 . Isotopic tracer in the expired 13 CO 2 / 12 CO 2 ratio was analyzed using a BreathMat Plus (Finnigan MAT). Exogenous glucose oxidation rate (EGO) was calculated using equation 3: (26; 39) (g?min -1 ) = V & CO 2 ? R OBS ? R REF ? 1 (Equation 3) R ING ? R REF k Where: R ? isotopic ratio, R OBS ? The observed 13 C/C ratio in the breath, R ING ? The 13 C/C ratio of the beverage ingested, R REF ? The baseline breath 13 C/C ratio, and k (0.747 L?g -1 ) is the amount of V & CO2 provide by the oxidation of 1 g of glucose. During the second hour of exercise, plasma samples were collected to allow for the calculation of exogenous glucose present in the blood, plasma glucose oxidation, liver glucose oxidation, and muscle glucose oxidation. These calculations were made only during the final 60 min of the 2-hour constant load ride allowing 60 min of equilibration. This 60 min equilibration is based on observations that 13 C/ 12 C in expired CO 2 equilibrates slowly with the 13 C/ 12 C in the CO 2 produced in tissues (38), that 13 C provided from 13 C-glucose is not irreversibly lost in tricarboxylic acid cycle intermediates (44) 56 and/or bicarbonate (48) pools, and that the 13 CO 2 recovery in expired gases is complete or almost complete. Plasma was deproteinized with barium hydroxide (0.3 N) and zinc sulfate (0.3 N). Glucose was then separated from the plasma by double-bed ion- exchange chromatography (AG 50W-X8 H + and AG 1-X8 chloride, 200-400 mesh; Bio- Rad, Mississauga, ON, Canada). The elute was then evaporated to dryness (Virtis Research Equipment, New York, NY) and combusted with copper oxide for 60 minutes at 400?C. The CO 2 was recovered from the glucose combustion for isotopic analysis. The measurement of 13 C/ 12 C in the CO 2 coming from combusted glucose was then performed using mass spectrometry (Prism, Manchester, UK). The isotopic ratio of the combusted plasma glucose was expressed in per mil difference by comparison with the PDB Chicago Standard (equation 4): ? [?- 13 C]PDB-1 = [R spl /R std ) ? 1] X 1,000 (Equation 4) Where: R spl ? 13 C-to- 12 C ratios in the sample and R std ? 13 C-to- 12 C ratios in the standard (1.1237 ?), respectively (4). The percentage of plasma glucose derived from exogenous glucose was calculated using equation 5: (%) = R GLU ? R REF ? 100 (Equation 5) R ING ? R REF 57 Where: R ? isotopic ratio, R GLU ? The observed 13 C/C ratio in the blood, R ING ? The 13 C/C ratio of the beverage ingested, and R REF ? The baseline blood 13 C/C ratio. Plasma glucose oxidation (PGO) was calculated using equation 6: (39) (g?min -1 ) = ____Plasma Glucose being Oxidized_____ (Equation 6) Exogenous Plasma Glucose Concentration Liver-derived glucose oxidation (LGO) was calculated using equation 7: (10; 18) (g?min -1 ) = PGO ? EGO (Equation 7) Muscle glycogen oxidation (expressed in grams of glucose/min) was calculated using equation 8: (10; 18) (g?min -1 ) = TCO - PGO (Equation 8) Statistical Analysis: All data are expressed as means ? standard errors. Statistical analysis was performed using SPSS version 13 (SPSS Inc. Chicago, IL). Data were analyzed with a univariate ANOVA. When ANOVA reported significant interactions, a Duncan post-hoc analysis was performed. Significance was set at p ? 0.05. 58 RESULTS Physiological Measures: V & O 2 increase significantly during the 2-hour constant load ride and was significantly higher in the 30 g?hr -1 and 60 g?hr -1 trials (Figure 3). This average V & O 2 for the four trials represented 77.2 ? 0.9% of the participants? V & O 2 PEAK . There was a significant reduction in RER (Figure 4) and carbohydrate oxidation (Figure 5) while fat oxidation (Figure 6) significantly increased during the 2-hour constant load ride (p ? 0.05). RER and carbohydrate oxidation were significantly higher while fat oxidation was significantly lower in the 60 g?hr -1 trial as compared to all other trials (p ? 0.05). Blood, Hormone, and Metabolite Measures: There were no differences in baseline blood glucose, insulin, or free-fatty acid measures. There were significant reductions in plasma glucose (Figure 7) and insulin (Figure 8) levels during the last hour of the 2-hour constant load ride. With reductions in carbohydrate ingestion below 30, plasma glucose levels declined at a significantly greater rate as carbohydrate ingestion declined. Plasma glucose levels were significantly lower in the PLA trial as compared to the 15 g?hr -1 trial. Plasma insulin levels were significantly higher in the 60 g?hr -1 trial as compared to all other trials. There was a significant rise in plasma free-fatty acid levels during the last hour of the 2-hour constant load ride. Plasma free-fatty acids increased during the second hour of the 2-hour constant load ride (Figure 9) There was no difference in plasma free-fatty acid levels when comparing the PLA and 15 g?hr -1 trials. As carbohydrate ingestion increased above 15, 59 serum free-fatty acid levels significantly increased as glucose ingestion increased (Figure 5). Exogenous and Endogenous Glucose Utilization: The expired 13 C/C ratio significantly increased during the 2-hour constant load ride with all treatments being significantly different at the beginning of the second hour of exercise (Figure 10). The percentage of exogenous glucose in the plasma increased significantly as exogenous carbohydrate intake increased (Figure 11). During the 2-hour constant load ride, exogenous glucose oxidation significantly increased as glucose ingestion rate increased. The areas under the curves for exogenous glucose, liver-derived glucose, and muscle glycogen were calculated for the second hour of the constant load ride (Figure 12). There was no difference in plasma glucose oxidation in the 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 trials. Since 13 C was not given in the PLA trial we are unable to compare plasma glucose oxidation in the PLA trial. Exogenous glucose oxidation rates during the second hour of the constant load cycling were 0.24 ? 0.01, 0.39 ? 0.01 and 0.58 ? 0.02 g?min -1 for 15 g?hr -1 , 30 g?hr -1 and 60 g?hr -1 ingestion rates, each being significantly different from each other (p ? 0.001). Exogenous glucose oxidation peaked in the final 30 minutes of the two hour constant load ride at 0.26 ? 0.05, 0.44 ? 0.04 and 0.66 ? 0.07 g?min -1 for 15 g?hr -1 , 30 g?hr -1 and 60 g?hr -1 trials, respectively (p ? 0.001). There was also a significant increase in liver glycogen oxidation during the last hour of the 2-hour constant load ride. Liver glucose oxidation rate was highest when consuming 15 g?hr -1 (0.84 ? 0.07 g?min -1 ) followed by 30 g?hr -1 (0.70 ? 0.07 g?min -1 ) and 60 g?hr -1 (0.56 ? 0.03 g?min -1 ), all significantly different from one another at a p ? 0.05 level. 60 There was also significant reduction in muscle glycogen oxidation during the last hour of the 2-hour constant load ride with no significant differences found between the 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 trials. No tracer was given in the PLA to examine muscle glycogen oxidation during the PLA trial. Performance Measures: The time required to complete the 20-km time trial was 36.39 ? 0.84 min, 35.23 ? 0.80 min, 34.99 ? 0.75 min, and 34.69 ? 0.62 min for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 , respectively. The change in time required to complete the 20-km time trial compared to PLA are shown in Figure 13. The average wattage during the time trial for PLA, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 was 212.0 ? 9.5 W, 227.9 ? 10.7 W, 229.7 ? 10.6 W, and 234.2 ? 9.4 W, respectively. PLA required significantly more time to complete the time trial and resulted in a significantly lower average wattage during the time trial compared to 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 (p ? 0.05). However, 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 were not significantly different from one another. DISCUSSION This study investigated the impact of glucose ingestion at rates of 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 on carbohydrate metabolism and exercise performance. The main findings of this investigation were: 1) exogenous glucose oxidation increased as glucose ingestion rate increased, 2) the ingestion of glucose at increasing rates provided increased protection of endogenous carbohydrate stores, and 3) ingesting glucose at rates equal to and greater than 15 g?hr -1 in improved cycling time-trial performance. 61 Many studies have examined the impact of carbohydrate ingestion on carbohydrate metabolism, substrate utilization, and exercise performance. This study has combined all three to determine the impact of carbohydrate on whole body carbohydrate metabolism, substrate utilization, and exercise performance. It has been demonstrated that exogenous carbohydrate quickly enters into the bloodstream providing an additional source of glucose (1; 11; 12; 50; 51; 58). The ingestion of carbohydrate has been reported to result in an elevation in plasma insulin as compared to the ingestion of water (27; 37). Elevations in circulating insulin and glucose promote increased glucose uptake by the exercising muscle (30; 58). In this investigation, plasma glucose was increased when ingesting carbohydrate at a rate of 30 g?hr -1 . Increasing rate to 60 g?hr -1 did not provide further elevations in plasma glucose. Interestingly, this study also demonstrated a significant rise in plasma insulin with a glucose ingestion rate of 60 g?hr -1 but no difference when comparing PLA, 15 g?hr -1 , and 30 g?hr -1 . Elevations in circulating insulin levels associated with glucose ingestion reduce the lipolytic rate and limit the availability of circulating free-fatty acids (56). Ingestion of carbohydrate and the impact on free-fatty acids response has previously been studied by Coyle?s laboratory (11; 12). We found circulating free-fatty acid levels during the 30 g?hr -1 trial were reduced as compared to the PLA and 15 g?hr -1 trial with the 60 g?hr -1 trial providing a further reduction. Costill et al (1977) demonstrated a sparing of muscle glycogen when plasma free-fatty acid levels were higher (9). It has been demonstrated that exogenous glucose oxidation rates rise as glucose intake rises until a threshold is reached (typically 1 g?min -1 ). The exogenous glucose oxidation rates found in this investigation are similar to those found in other 62 investigations (26; 54). This study suggests the percentage of exogenous glucose being oxidized is influenced by ingestion rate. Exogenous oxidation rates peaked during the last 30 minutes of the two-hour constant load ride. The peak percentage of exogenous glucose oxidation was 104%, 88%, and 66% of the ingestion rate during the 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 , respectively. The reason peak glucose oxidation rate in the 15 g?hr -1 exceeds the ingestion rate may be explained by the oxidation of glucose ingested earlier in the trial since peak rates were observed during the last 30 minutes of each trial. Wallis et al (2007) reported the percentage of exogenous glucose being oxidized with ingestion rates of 30 g?hr -1 and 60 g?hr -1 was 66% and 50%, respectively in women (54). They suggested that some of the carbohydrate that is ingested may remain in the intestines or may go to the liver or inactive muscles to be stored (26; 54). With exogenous glucose oxidation rates in the 30 g?hr -1 exceeding ingestion rates in the 15 g?hr -1 , as well as exogenous glucose oxidation rates in the 60 g?hr -1 exceeding ingestion rates in the 30 g?hr -1 , it seems intestinal absorption is not a major limiting factor for the oxidation of exogenous glucose at rates below 60 g?hr -1 . Several studies have also demonstrated that exogenous carbohydrate oxidation rates peak at approximately 1.0 g?min -1 when a single form of carbohydrate is ingested in large amounts (25; 26; 53). Therefore, ingestion rates of 60 g?hr -1 (1.0 g?min -1 ) are likely able to be absorbed by the intestine at a rate allowing most of the ingested carbohydrate to be quickly oxidized. This suggests that when glucose is ingested at greater than 15 g?hr -1 rates the difference in ingestion rate and exogenous oxidation rate is likely being taken-up or stored by the liver and muscle. It seems that when exogenous glucose is quickly made available to the exercising muscle, the demand to utilize endogenous stores of carbohydrate for energy is not as 63 great (20; 22; 23; 27; 37; 43). Although, there continues to be some debate as to what endogenous carbohydrate sources are spared with carbohydrate ingestion. The sparing of liver-derived glucose when consuming carbohydrate has been repeatedly demonstrated (26; 27; 30; 39; 54). In agreement with this we demonstrated a reduction in the liver- derived glucose oxidation during the last 30 minutes of exercise as carbohydrate ingestion rate increased. Several studies using running and variable intensity cycling as the exercise modes have demonstrated muscle glycogen sparing (37; 50; 51; 58). Constant load cycling studies have failed to demonstrate a sparing of muscle glycogen stores with exogenous carbohydrate (7; 13; 54). Since 13 C was not added to the placebo treatment in this study, no comparison can be made concerning the changes in muscle glycogen oxidation between carbohydrate ingestion trials and the placebo trial. However, increasing the rate of glucose ingestion did not alter the rate of muscle glycogen oxidation. The alterations of available glucose, insulin, and free-fatty acids, seen with the three glucose treatments, may have led to the same amount of fuel supplied to the working muscle, resulting in observation of similar rates of muscle glycogen oxidation. The impact of carbohydrate as an ergogenic aid in exercise lasting longer than 1- hour is well accepted. Many researchers have demonstrated that carbohydrate ingestion improves exercise performance as compared to not receiving carbohydrate (1; 11; 16; 17; 29; 32; 33; 57; 58). Several studies have investigated the impact of increasing carbohydrate intake rate on exercise performance. It would seem that large carbohydrate ingestion rates would provide the exercising muscle more fuel to continue work. Mitchell et al found carbohydrate ingestion rates, ranging from 34 to 111 g?hr -1 , did not vary in their impact on exercise performance (32; 33). Gastric emptying rates (31; 34) 64 and intestinal absorption rates (41) have been identified as potential reasons large carbohydrate intakes during exercise do not provide additional performance benefits. While many have tried to maximize exercise performance by increasing carbohydrate ingestion rate, fewer studies tried to determine the minimal amount of carbohydrate needed to elicit performance benefits. There have been several studies suggesting performance can be enhanced with carbohydrate ingestion rates near 30 g?hr -1 (33; 35; 36; 47; 55) and other studies have demonstrated carbohydrate ingestion below 30 g?hr -1 can elicit performance benefits (15; 16; 28; 29). This study found carbohydrate ingestion rates ranging from 15 g?hr -1 to 60 g?hr -1 provide performance benefits. In the 60 g?hr -1 trial all twelve subjects completed the simulated time-trial faster as compared to the PLA trial. When ingesting glucose at rates of 15 g?hr -1 and 30 g?hr -1 , nine subjects finished the time trial faster than PLA trial. Exogenous carbohydrate oxidation plateaus when exercise intensity exceeds 50% V & O 2 max (40). Since there are only limited stores of carbohydrate, the body must utilize carbohydrate from external sources or reduce intensity to continue work. As the carbohydrate available to the exercising muscle declines during prolonged exercise, the utilization of fat for energy increases. As fat?s role in energy production increases, work intensity declines due to the slower rate of ATP production from fat metabolism. Exogenous carbohydrate?s incorporation into the bloodstream provides another source of carbohydrate which allows for maintained work output and spares some of the hepatic stores of carbohydrate. These findings as well as those of many other researchers demonstrate the ability of carbohydrate ingestion to sustain work and improve performance. In 1987, Coggan 65 and Coyle suggested that fatigue during cycling was due to an insufficient supply of carbohydrate reaching the exercising muscle (6). This data adds to the literature demonstrating athletes can maintain higher exercise intensities and improve exercise performance if exogenous carbohydrate sources maintain blood glucose and reduce the body?s reliance on endogenous carbohydrate sources for energy. In summary, this study found carbohydrate ingestion increased plasma glucose with ingestion rates as low as 15 g?hr -1 . Ingesting glucose at 30 and 60 g?hr -1 resulted in an increase in plasma glucose greater than what was observed when ingesting 15 g?hr -1 . Plasma insulin did not demonstrate a significant elevation until carbohydrate ingestion reached 60 g?hr -1 . A reduction in serum free-fatty acid was observed when glucose ingestion reached 30 g?hr -1 with a further reduction during the 60 g?hr -1 trial. Exogenous carbohydrate oxidation rates increased as carbohydrate ingestion rate increased from 15 g?hr -1 to 30 g?hr -1 , with a further increase observed during the 60 g?hr -1 trial. As glucose ingestion rate increased a smaller percentage of exogenous glucose was oxidized. Liver- derived glucose oxidation was reduced as glucose ingestion rate increased. Glucose ingestion did not alter muscle glycogen oxidation. Glucose ingestion resulted in improved time-trial performance but alterations in carbohydrate dose did not impact the performance benefit. In conclusion, the findings of this study indicated that increasing glucose ingestion rate increases oxidation of exogenous glucose and spares liver-derived glucose. This study also demonstrates that 60 g?hr -1 provided the greatest protection on liver-derived glucose and resulted in all subjects completing the time trial faster than placebo, even though performance was improved in nine of the twelve subjects when ingesting glucose at a rate as low as 15 g?hr -1 during exercise lasting approximately 150 66 minutes. Future research is needed to explore the potential benefits and drawbacks of ingesting carbohydrate at rates lower than 30-60 g?hr -1 . REFERENCES 1. Bacharach DW, von Duvillard SP, Rundell KW, Meng J, Cring MR, Szmedra L and Castle JM. Carbohydrate drinks and cycling performance. J Sports Med Phys Fitness 34: 161-168, 1994. 2. Below PR, Mora-Rodriguez R, Gonzalez-Alonso J and Coyle EF. Fluid and carbohydrate ingestion independently improve performance during 1 h of intense exercise. Med Sci Sports Exerc 27: 200-210, 1995. 3. Bosch AN, Dennis SC and Noakes TD. Influence of carbohydrate ingestion on fuel substrate turnover and oxidation during prolonged exercise. J Appl Physiol 76: 2364-2372, 1994. 4. Burelle Y, Peronnet F, Charpentier S, Lavoie C, Hillaire-Marcel C and Massicotte D. Oxidation of an oral [13C]glucose load at rest and prolonged exercise in trained and sedentary subjects. J Appl Physiol 86: 52-60, 1999. 5. Casa DJ, Armstrong LE, Hillman SK, Montain SJ, Reiff RV, Rich BS, Roberts WO and Stone JA. National Athletic Trainers' Association Position Statement: Fluid Replacement for Athletes. J Athl Train 35: 212-224, 2000. 67 6. Coggan AR and Coyle EF. Reversal of fatigue during prolonged exercise by carbohydrate infusion or ingestion. J Appl Physiol 63: 2388-2395, 1987. 7. Coggan AR and Coyle EF. Carbohydrate ingestion during prolonged exercise: effects on metabolism and performance. Exerc Sport Sci Rev 19: 1-40, 1991. 8. Coombes JS and Hamilton KL. The effectiveness of commercially available sports drinks. Sports Med 29: 181-209, 2000. 9. Costill DL, Coyle E, Dalsky G, Evans W, Fink W and Hoopes D. Effects of elevated plasma FFA and insulin on muscle glycogen usage during exercise. J Appl Physiol 43: 695-699, 1977. 10. Couture S, Massicotte D, Lavoie C, Hillaire-Marcel C and Peronnet F. Oral [(13)C]glucose and endogenous energy substrate oxidation during prolonged treadmill running. J Appl Physiol 92: 1255-1260, 2002. 11. Coyle EF, Coggan AR, Hemmert MK and Ivy JL. Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate. J Appl Physiol 61: 165-172, 1986. 12. Coyle EF, Hagberg JM, Hurley BF, Martin WH, Ehsani AA and Holloszy JO. Carbohydrate feeding during prolonged strenuous exercise can delay fatigue. J Appl Physiol 55: 230-235, 1983. 68 13. Coyle EF, Hamilton MT, Alonso JG, Montain SJ and Ivy JL. Carbohydrate metabolism during intense exercise when hyperglycemic. J Appl Physiol 70: 834- 840, 1991. 14. Davis JM, Burgess WA, Slentz CA, Bartoli WP and Pate RR. Effects of ingesting 6% and 12% glucose/electrolyte beverages during prolonged intermittent cycling in the heat. Eur J Appl Physiol Occup Physiol 57: 563-569, 1988. 15. el-Sayed MS, Balmer J and Rattu AJ. Carbohydrate ingestion improves endurance performance during a 1 h simulated cycling time trial. J Sports Sci 15: 223-230, 1997. 16. Fielding RA, Costill DL, Fink WJ, King DS, Hargreaves M and Kovaleski JE. Effect of carbohydrate feeding frequencies and dosage on muscle glycogen use during exercise. Med Sci Sports Exerc 17: 472-476, 1985. 17. Hargreaves M, Costill DL, Coggan A, Fink WJ and Nishibata I. Effect of carbohydrate feedings on muscle glycogen utilization and exercise performance. Med Sci Sports Exerc 16: 219-222, 1984. 18. Harvey CR, Frew R, Massicotte D, Peronnet F and Rehrer NJ. Muscle glycogen oxidation during prolonged exercise measured with oral [13C]glucose: comparison with changes in muscle glycogen content. J Appl Physiol 102: 1773- 1779, 2007. 69 19. Hermansen L, Hultman E and Saltin B. Muscle glycogen during prolonged severe exercise. Acta Physiol Scand 71: 129-139, 1967. 20. Jentjens RL and Jeukendrup AE. High rates of exogenous carbohydrate oxidation from a mixture of glucose and fructose ingested during prolonged cycling exercise. Br J Nutr 93: 485-492, 2005. 21. Jentjens RL, Moseley L, Waring RH, Harding LK and Jeukendrup AE. Oxidation of combined ingestion of glucose and fructose during exercise. J Appl Physiol 96: 1277-1284, 2004. 22. Jentjens RL, Shaw C, Birtles T, Waring RH, Harding LK and Jeukendrup AE. Oxidation of combined ingestion of glucose and sucrose during exercise. Metabolism 54: 610-618, 2005. 23. Jentjens RL, Venables MC and Jeukendrup AE. Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise. J Appl Physiol 96: 1285- 1291, 2004. 24. Jeukendrup AE. Carbohydrate intake during exercise and performance. Nutrition 20: 669-677, 2004. 25. Jeukendrup AE, Borghouts LB, Saris WH and Wagenmakers AJ. Reduced oxidation rates of ingested glucose during prolonged exercise with low endogenous CHO availability. J Appl Physiol 81: 1952-1957, 1996. 70 26. Jeukendrup AE, Wagenmakers AJ, Stegen JH, Gijsen AP, Brouns F and Saris WH. Carbohydrate ingestion can completely suppress endogenous glucose production during exercise. Am J Physiol 276: E672-E683, 1999. 27. Massicotte D, Peronnet F, Allah C, Hillaire-Marcel C, Ledoux M and Brisson G. Metabolic response to [13C]glucose and [13C]fructose ingestion during exercise. J Appl Physiol 61: 1180-1184, 1986. 28. Maughan RJ, Bethell LR and Leiper JB. Effects of ingested fluids on exercise capacity and on cardiovascular and metabolic responses to prolonged exercise in man. Exp Physiol 81: 847-859, 1996. 29. Maughan RJ, Fenn CE and Leiper JB. Effects of fluid, electrolyte and substrate ingestion on endurance capacity. Eur J Appl Physiol Occup Physiol 58: 481-486, 1989. 30. McConell G, Fabris S, Proietto J and Hargreaves M. Effect of carbohydrate ingestion on glucose kinetics during exercise. J Appl Physiol 77: 1537-1541, 1994. 31. McHugh PR and Moran TH. Calories and gastric emptying: a regulatory capacity with implications for feeding. Am J Physiol 236: R254-R260, 1979. 32. Mitchell JB, Costill DL, Houmard JA, Fink WJ, Pascoe DD and Pearson DR. Influence of carbohydrate dosage on exercise performance and glycogen metabolism. J Appl Physiol 67: 1843-1849, 1989. 71 33. Mitchell JB, Costill DL, Houmard JA, Flynn MG, Fink WJ and Beltz JD. Effects of carbohydrate ingestion on gastric emptying and exercise performance. Med Sci Sports Exerc 20: 110-115, 1988. 34. Moran TH and McHugh PR. Distinctions among three sugars in their effects on gastric emptying and satiety. Am J Physiol 241: R25-R30, 1981. 35. Murray R, Paul GL, Seifert JG and Eddy DE. Responses to varying rates of carbohydrate ingestion during exercise. Med Sci Sports Exerc 23: 713-718, 1991. 36. Murray R, Seifert JG, Eddy DE, Paul GL and Halaby GA. Carbohydrate feeding and exercise: effect of beverage carbohydrate content. Eur J Appl Physiol Occup Physiol 59: 152-158, 1989. 37. Nicholas CW, Tsintzas K, Boobis L and Williams C. Carbohydrate-electrolyte ingestion during intermittent high-intensity running. Med Sci Sports Exerc 31: 1280-1286, 1999. 38. Pallikarakis N, Sphiris N and Lefebvre P. Influence of the bicarbonate pool and on the occurrence of 13CO2 in exhaled air. Eur J Appl Physiol Occup Physiol 63: 179-183, 1991. 39. Peronnet F, Rheaume N, Lavoie C, Hillaire-Marcel C and Massicotte D. Oral [13C]glucose oxidation during prolonged exercise after high- and low- carbohydrate diets. J Appl Physiol 85: 723-730, 1998. 72 40. Pirnay F, Crielaard JM, Pallikarakis N, Lacroix M, Mosora F, Krzentowski G, Luyckx AS and Lefebvre PJ. Fate of exogenous glucose during exercise of different intensities in humans. J Appl Physiol 53: 1620-1624, 1982. 41. Radziuk J and Bondy DC. Abnormal oral glucose tolerance and glucose malabsorption after vagotomy and pyloroplasty. A tracer method for measuring glucose absorption rates. Gastroenterology 83: 1017-1025, 1982. 42. Rehrer NJ, Wagenmakers AJ, Beckers EJ, Halliday D, Leiper JB, Brouns F, Maughan RJ, Westerterp K and Saris WH. Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise. J Appl Physiol 72: 468-475, 1992. 43. Riddell MC, Bar-Or O, Schwarcz HP and Heigenhauser GJ. Substrate utilization in boys during exercise with [13C]-glucose ingestion. Eur J Appl Physiol 83: 441- 448, 2000. 44. Ruzzin J, Peronnet F, Tremblay J, Massicotte D and Lavoie C. Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose. J Appl Physiol 95: 477-482, 2003. 45. Ryan AJ, Lambert GP, Shi X, Chang RT, Summers RW and Gisolfi CV. Effect of hypohydration on gastric emptying and intestinal absorption during exercise. J Appl Physiol 84: 1581-1588, 1998. 73 46. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ and Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports Exerc 39: 377-390, 2007. 47. Sugiura K and Kobayashi K. Effect of carbohydrate ingestion on sprint performance following continuous and intermittent exercise. Med Sci Sports Exerc 30: 1624-1630, 1998. 48. Trimmer JK, Casazza GA, Horning MA and Brooks GA. Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions. Am J Physiol Endocrinol Metab 281: E683-E692, 2001. 49. Tsintzas K and Williams C. Human muscle glycogen metabolism during exercise. Effect of carbohydrate supplementation. Sports Med 25: 7-23, 1998. 50. Tsintzas OK, Williams C, Boobis L and Greenhaff P. Carbohydrate ingestion and glycogen utilization in different muscle fibre types in man. J Physiol 489 ( Pt 1): 243-250, 1995. 51. Tsintzas OK, Williams C, Boobis L and Greenhaff P. Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men. J Appl Physiol 81: 801-809, 1996. 52. Vist GE and Maughan RJ. Gastric emptying of ingested solutions in man: effect of beverage glucose concentration. Med Sci Sports Exerc 26: 1269-1273, 1994. 74 53. Wagenmakers AJ, Brouns F, Saris WH and Halliday D. Oxidation rates of orally ingested carbohydrates during prolonged exercise in men. J Appl Physiol 75: 2774-2780, 1993. 54. Wallis GA, Yeo SE, Blannin AK and Jeukendrup AE. Dose-response effects of ingested carbohydrate on exercise metabolism in women. Med Sci Sports Exerc 39: 131-138, 2007. 55. Williams C, Nute MG, Broadbank L and Vinall S. Influence of fluid intake on endurance running performance. Eur J Appl Physiol 60: 112-119, 1990. 56. Wolfe RR, Nadel ER, Shaw JH, Stephenson LA and Wolfe MH. Role of changes in insulin and glucagon in glucose homeostasis in exercise. J Clin Invest 77: 900- 907, 1986. 57. Wright DA, Sherman WM and Dernbach AR. Carbohydrate feedings before, during, or in combination improve cycling endurance performance. J Appl Physiol 71: 1082-1088, 1991. 58. Yaspelkis BB, III, Patterson JG, Anderla PA, Ding Z and Ivy JL. Carbohydrate supplementation spares muscle glycogen during variable-intensity exercise. J Appl Physiol 75: 1477-1485, 1993. 75 Figure 1: Order of test measures Diagram of the testing protocol. 0 15 30 45 60 75 90 105 120 RPE RPE RPE RPE RPE RPE RPE RPE HR HR HR HR HR HR HR HR V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 V & O 2 Blood Blood Blood Blood Blood Blood Drink Drink Drink Drink Drink Drink Drink Drink Constant Load Ride 20-K TT 76 Figure 2: 20-kilometer course profile Course profile of the 20-km time trial. 77 Figure 3 ? V & O 2 during exercise 2.50 2.75 3.00 3.25 3.50 15 30 45 60 75 90 105 120 Minutes L?min -1 PLA 15 g/min 30 g/min 60 g/min Oxygen uptake response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA and 60 g?hr -1 . b significantly higher than 60 g?hr -1 . There is a significant main effect for time (p < 0.05). a b 78 Figure 4 ? Respiratory exchange ratio during exercise 0.85 0.90 0.95 1.00 15 30 45 60 75 90 105 120 Minutes RER PLA 15 g/min 30 g/min 60 g/min Respiratory exchange ratio response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA, 15 g?hr -1 , and 30 g?hr -1 There is a significant main effect for time (p < 0.05). a 79 Figure 5 ? 13 C/C ratio in expired air during exercise -30 -25 -20 -15 -10 -5 0 5 10 15 0 153045607590105120 Minutes ? [ ? -13C]PDB -1 PLA 15 g/hr 30 g/hr 60 g/hr Expired PDB in the breath during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a 15 g?min -1 significantly lower than PLA, 30 g?min -1 and 60 g?min -1 , b PLA significantly lower than 15 g?min -1 , 30 g?min -1 , and 60 g?min -1 , c 15 g?min -1 significantly lower than 30 g?min -1 and 60 g?min -1 , and d 30 g?min -1 significantly lower than 60 g?min -1 . a bc bcd bcd bcd bcd bcd 80 Figure 6 ? Carbohydrate oxidation during exercise 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 15 30 45 60 75 90 105 120 Minutes g?min -1 PLA 15 g/min 30 g/min 60 g/min Carbohydrate oxidation response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than PLA, 15 g?hr -1 ,and 30 g?hr -1 . There is a significant main effect for time (p < 0.05). a 81 Figure 7 ? Fat oxidation during exercise 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 15 30 45 60 75 90 105 120 Minutes g/min PLA 15 g/hr 30 g/hr 60 g/hr Fat oxidatoion during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly lower than PLA, 15 g?hr -1 ,and 30 g?hr -1 . There is a significant main effect for time (p < 0.05). a 82 Figure 8 ? Blood glucose response during exercise 60 70 80 90 100 110 120 60 75 90 105 120 Minutes mg?dL -1 PLA 15 g/min 30 g/min 60 g/min Plasma glucose response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than 0; b significantly higher than 15 g?hr -1 . There is a significant main effect for time (p < 0.05). ab ab a 83 Figure 9 ? Insulin response during exercise 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 60 75 90 105 120 Minutes mU?L -1 PLA 15 g/min 30 g/min 60 g/min Plasma insulin response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly higher than all other treatments (p < 0.05). There is a significant main effect for time (p < 0.05). a 84 Figure 10 ? Free fatty acid response during exercise 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 60 75 90 105 120 Minutes mE q?L -1 PLA 15 g/min 30 g/min 60 g/min Serum free-fatty acid response during a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). a significantly lower than 0 and 15; b significantly lower 30. (p < 0.05). There is a significant main effect for time (p < 0.05). a ab 85 Figure 11 ? Percent plasma glucose coming from an exogenous source 0 10 20 30 40 50 60 70 80 90 100 PLA 15 g/min 30 g/min 60 g/min Trial % 60 min 90 min 120 min Percent plasma glucose coming from an exogenous source during the last hour of a 2- hour ride at 95% of the workload that elicits a 4 mmol blood lactate response. Values are means (SE). All trials are significantly different from each other. There were no significant differences in regards to time within trials. 86 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 Time 60 90 120 60 90 1 20 60 90 120 minute s Tr e a t men t 15 g ? h -1 30 g ? h -1 60 g ? h -1 Musc le g l y c o gen L i ve r Gluc os e Ex og e nous Gluc ose 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 g? m i n -1 F i g u r e 12 ? S ou r c e of ox idiz e d c a r boh y d ra t e duri n g th e l a st hour of e x e r c i se whe n c onsu m in g g l u c o s e a t 15, 30 , a nd 60 g ? h -1 . Car boh y d rat e utiliz a tion during the fin a l hour of a 2-hour ride a t 9 5 % of th e worklo a d th a t e l i c its a 4 mmol bloo d lac t a t e r e s p o nse whe n c onsu m in g 15, 3 0, a nd 60 g of g l u c ose an h our. Va lu e s a r e m e a n s ( S E) . a sig n if ic antl y l e ss th an 15 g ? h -1 ; b s i g n i f ic a n tly le ss tha n 30 g ? h -1 ; c sig n i f ic antl y gre a t e r tha n 15 g?h -1 ; d si g n ifica n tl y g r ea t e r t h a n 30 g ? h -1 . (p < 0.0 5) a c ab cd 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 4. 0 Time 60 90 120 60 90 1 20 60 90 120 minute s Tr e a t men t 15 g ? h -1 30 g ? h -1 60 g ? h -1 Musc le g l y c o gen L i ve r Gluc os e Ex og e nous Gluc ose 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 g? m i n -1 F i g u r e 12 ? S ou r c e of ox idiz e d c a r boh y d ra t e duri n g th e l a st hour of e x e r c i se whe n c onsu m in g g l u c o s e a t 15, 30 , a nd 60 g ? h -1 . Car boh y d rat e utiliz a tion during the fin a l hour of a 2-hour ride a t 9 5 % of th e worklo a d th a t e l i c its a 4 mmol bloo d lac t a t e r e s p o nse whe n c onsu m in g 15, 3 0, a nd 60 g of g l u c ose an h our. Va lu e s a r e m e a n s ( S E) . a sig n if ic antl y l e ss th an 15 g ? h -1 ; b s i g n i f ic a n tly le ss tha n 30 g ? h -1 ; c sig n i f ic antl y gre a t e r tha n 15 g?h -1 ; d si g n ifica n tl y g r ea t e r t h a n 30 g ? h -1 . (p < 0.0 5) a c ab cd 87 Figure 13: Change in 20-km time trial completion time in relation to PLA -7 -6 -5 -4 -3 -2 -1 0 1 2 3 15 30 60 Treatment Minutes Box plots of change in duration in the 20-km time trial performance following a 2-hour ride at 95% of the workload that elicits a 4 mmol blood lactate response when consuming glucose at various rates. The top and bottom of the box represents the 75 th and 25 th percentile. The whiskers capture the range of performance times for the entire group of subjects. The black line in the box corresponds to the median performance value. No differences were observed between glucose treatments. All treatments were significantly faster than placebo. 88 VI: SUMMARY This study examined the physiological and exercise performance responses of cyclists during a 2-hour constant load ride followed by a simulated 20-km time trial to determine the impact of beverages with glucose concentrations at and below what is recommended by the American College of Sports Medicine and the National Athletic Trainer Association. Participants completed four trials including an electrolyte containing placebo beverage trial with no carbohydrate and three trials during which a glucose/electrolyte beverage was ingested at a rate of 15 g?h -1 , 30 g?h -1 , and 60 g?h -1 . This study investigated the impact of glucose ingestion at rates of 15 g?hr -1 , 30 g?hr -1 , and 60 g?hr -1 on carbohydrate metabolism and exercise performance. The main findings of this investigation were: 1) exogenous glucose oxidation increased as glucose ingestion rate increased, 2) the ingestion of glucose at increasing rates provides increased protection of liver glucose stores, and 3) ingesting glucose at rates equal to and greater than 15 g?h -1 result in improved cycling time-trial performance. Exogenous glucose oxidation rates increased as glucose ingestion rates increased. This demonstrates that the glucose ingested during exercise can be readily used for energy. With exogenous glucose oxidation rates in the 30 g?hr -1 exceeding ingestion rates in the 15 g?hr -1 , as well as exogenous glucose oxidation rates in the 60 g?hr -1 exceeding ingestion rates in the 30 g?hr -1 , it seems that intestinal absorption is not a major limiting factor to the oxidation of exogenous glucose at low ingestion rates. 89 As glucose ingestion increased the reliance on muscle glycogen did not change but the utilization of liver-derived glucose for energy was reduced. Delaying the utilization of endogenous sources of carbohydrate would provide the body greater energy later in exercise allowing the maintenance of workloads further into the duration of exercise. Glucose ingestion did not alter heart rate, ratings of perceived exertion, respiratory exchange ratio, blood lactate, or cortisol. Blood glucose levels were significantly elevated compared to placebo with glucose ingestion rates as low as 15 g?hr - 1 and were further elevated with ingestion rates of 30 and 60 g?hr -1 . Blood insulin levels were significantly elevated compared to placebo only when glucose was ingested at 60 g?hr -1 . Free-fatty acid levels were reduced with a glucose ingestion rate of 30 g?hr -1 and further reduced with a glucose ingestion rate of 60 g?hr -1 . Time to complete the 20-km time-trial and average wattages of the 20-km time- trial were significantly improved with a glucose ingestion rate of 15 g?hr -1 as compared to placebo. Increasing glucose ingestion rate provided no further improvements. Potential areas for future research in this area include: 1) determining the impact of other carbohydrate and fat fuel source on physiological and performance measures; 2) exploring the impact multiple carbohydrate sources delivered in reduced carbohydrate beverages on physiological and performance measures; 3) exploring the mechanism that allows for similar improvements in exercise performance with variations in the utilization of exogenous and endogenous energy sources; and 4) exploring methods to maximize endogenous and exogenous substrate utilization to enhance exercise performance. 90 REFERENCES 1. Ali A, Williams C, Nicholas CW and Foskett A. The influence of carbohydrate- electrolyte ingestion on soccer skill performance. Med Sci Sports Exerc 39: 1969- 1976, 2007. 2. Anantaraman R, Carmines AA, Gaesser GA and Weltman A. Effects of carbohydrate supplementation on performance during 1 hour of high-intensity exercise. Int J Sports Med 16: 461-465, 1995. 3. Andrews JL, Sedlock DA, Flynn MG, Navalta JW and Ji H. Carbohydrate loading and supplementation in endurance-trained women runners. J Appl Physiol 95: 584-590, 2003. 4. Bacharach DW, von Duvillard SP, Rundell KW, Meng J, Cring MR, Szmedra L and Castle JM. Carbohydrate drinks and cycling performance. J Sports Med Phys Fitness 34: 161-168, 1994. 5. Bagby GJ, Green HJ, Katsuta S and Gollnick PD. Glycogen depletion in exercising rats infused with glucose, lactate, or pyruvate. J Appl Physiol 45: 425- 429, 1978. 91 6. Ball TC, Headley SA, Vanderburgh PM and Smith JC. Periodic carbohydrate replacement during 50 min of high-intensity cycling improves subsequent sprint performance. Int J Sport Nutr 5: 151-158, 1995. 7. Barnes G, Morton A and Wilson A. The effect of new glucose-electrolyte fluid on blood electrolyte levels, gastric emptying and work performance. Aust J Sci Med Sport 16: 25-30, 1984. 8. Below PR, Mora-Rodriguez R, Gonzalez-Alonso J and Coyle EF. Fluid and carbohydrate ingestion independently improve performance during 1 h of intense exercise. Med Sci Sports Exerc 27: 200-210, 1995. 9. Benade AJ, Wyndham CH, Jansen CR, Rogers GG and de Bruin EJ. Plasma insulin and carbohydrate metabolism after sucrose ingestion during rest and prolonged aerobic exercise. Pflugers Arch 342: 207-218, 1973. 10. Bergstrom J, Hermansen L, Hultman E and Saltin B. Diet, muscle glycogen and physical performance. Acta Physiol Scand 71: 140-150, 1967. 11. Bergstrom J and Hultman E. A study of the glycogen metabolism during exercise in man. Scand J Clin Lab Invest 19: 218-228, 1967. 12. Bjorkman O, Sahlin K, Hagenfeldt L and Wahren J. Influence of glucose and fructose ingestion on the capacity for long-term exercise in well-trained men. Clin Physiol 4: 483-494, 1984. 92 13. Bonen A, Malcolm SA, Kilgour RD, MacIntyre KP and Belcastro AN. Glucose ingestion before and during intense exercise. J Appl Physiol 50: 766-771, 1981. 14. Bosch AN, Dennis SC and Noakes TD. Influence of carbohydrate ingestion on fuel substrate turnover and oxidation during prolonged exercise. J Appl Physiol 76: 2364-2372, 1994. 15. Burant CF, Takeda J, Brot-Laroche E, Bell GI and Davidson NO. Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem 267: 14523-14526, 1992. 16. Burelle Y, Peronnet F, Charpentier S, Lavoie C, Hillaire-Marcel C and Massicotte D. Oxidation of an oral [13C]glucose load at rest and prolonged exercise in trained and sedentary subjects. J Appl Physiol 86: 52-60, 1999. 17. Burgess WA, Davis JM, Bartoli WP and Woods JA. Failure of low dose carbohydrate feeding to attenuate glucoregulatory hormone responses and improve endurance performance. Int J Sport Nutr 1: 338-352, 1991. 18. Casa DJ, Armstrong LE, Hillman SK, Montain SJ, Reiff RV, Rich BS, Roberts WO and Stone JA. National Athletic Trainers' Association Position Statement: Fluid Replacement for Athletes. J Athl Train 35: 212-224, 2000. 19. Chryssanthopoulos C and Williams C. Pre-exercise carbohydrate meal and endurance running capacity when carbohydrates are ingested during exercise. Int J Sports Med 18: 543-548, 1997. 93 20. Coggan AR and Coyle EF. Reversal of fatigue during prolonged exercise by carbohydrate infusion or ingestion. J Appl Physiol 63: 2388-2395, 1987. 21. Coggan AR and Coyle EF. Effect of carbohydrate feedings during high-intensity exercise. J Appl Physiol 65: 1703-1709, 1988. 22. Coggan AR and Coyle EF. Carbohydrate ingestion during prolonged exercise: effects on metabolism and performance. Exerc Sport Sci Rev 19: 1-40, 1991. 23. Coggan AR, Habash DL, Mendenhall LA, Swanson SC and Kien CL. Isotopic estimation of CO2 production during exercise before and after endurance training. J Appl Physiol 75: 70-75, 1993. 24. Coombes JS and Hamilton KL. The effectiveness of commercially available sports drinks. Sports Med 29: 181-209, 2000. 25. Costill DL. Carbohydrate nutrition before, during, and after exercise. Fed Proc 44: 364-368, 1985. 26. Costill DL, Coyle E, Dalsky G, Evans W, Fink W and Hoopes D. Effects of elevated plasma FFA and insulin on muscle glycogen usage during exercise. J Appl Physiol 43: 695-699, 1977. 27. Costill DL and Saltin B. Factors limiting gastric emptying during rest and exercise. J Appl Physiol 37: 679-683, 1974. 94 28. Couture S, Massicotte D, Lavoie C, Hillaire-Marcel C and Peronnet F. Oral [(13)C]glucose and endogenous energy substrate oxidation during prolonged treadmill running. J Appl Physiol 92: 1255-1260, 2002. 29. Coyle EF, Coggan AR, Hemmert MK and Ivy JL. Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate. J Appl Physiol 61: 165-172, 1986. 30. Coyle EF, Costill DL, Fink WJ and Hoopes DG. Gastric emptying rates for selected athletic drinks. Res Q 49: 119-124, 1978. 31. Coyle EF, Hagberg JM, Hurley BF, Martin WH, Ehsani AA and Holloszy JO. Carbohydrate feeding during prolonged strenuous exercise can delay fatigue. J Appl Physiol 55: 230-235, 1983. 32. Coyle EF, Hamilton MT, Alonso JG, Montain SJ and Ivy JL. Carbohydrate metabolism during intense exercise when hyperglycemic. J Appl Physiol 70: 834- 840, 1991. 33. Davis JM, Burgess WA, Slentz CA, Bartoli WP and Pate RR. Effects of ingesting 6% and 12% glucose/electrolyte beverages during prolonged intermittent cycling in the heat. Eur J Appl Physiol Occup Physiol 57: 563-569, 1988. 34. Davis JM, Jackson DA, Broadwell MS, Queary JL and Lambert CL. Carbohydrate drinks delay fatigue during intermittent, high-intensity cycling in active men and women. Int J Sport Nutr 7: 261-273, 1997. 95 35. Decombaz J, Arnaud MJ, Milon H, Moesch H, Philippossian G, Thelin AL and Howald H. Energy metabolism of medium-chain triglycerides versus carbohydrates during exercise. Eur J Appl Physiol Occup Physiol 52: 9-14, 1983. 36. el-Sayed MS, Balmer J and Rattu AJ. Carbohydrate ingestion improves endurance performance during a 1 h simulated cycling time trial. J Sports Sci 15: 223-230, 1997. 37. el-Sayed MS, Rattu AJ and Roberts I. Effects of carbohydrate feeding before and during prolonged exercise on subsequent maximal exercise performance capacity. Int J Sport Nutr 5: 215-224, 1995. 38. Erickson MA, Schwarzkopf RJ and McKenzie RD. Effects of caffeine, fructose, and glucose ingestion on muscle glycogen utilization during exercise. Med Sci Sports Exerc 19: 579-583, 1987. 39. Febbraio MA, Chiu A, Angus DJ, Arkinstall MJ and Hawley JA. Effects of carbohydrate ingestion before and during exercise on glucose kinetics and performance. J Appl Physiol 89: 2220-2226, 2000. 40. Felig P and Wahren J. Fuel homeostasis in exercise. N Engl J Med 293: 1078- 1084, 1975. 41. Ferraris RP and Diamond J. Regulation of intestinal sugar transport. Physiol Rev 77: 257-302, 1997. 96 42. Fielding RA, Costill DL, Fink WJ, King DS, Hargreaves M and Kovaleski JE. Effect of carbohydrate feeding frequencies and dosage on muscle glycogen use during exercise. Med Sci Sports Exerc 17: 472-476, 1985. 43. Flynn MG, Costill DL, Hawley JA, Fink WJ, Neufer PD, Fielding RA and Sleeper MD. Influence of selected carbohydrate drinks on cycling performance and glycogen use. Med Sci Sports Exerc 19: 37-40, 1987. 44. Fordtran JS and Saltin B. Gastric emptying and intestinal absorption during prolonged severe exercise. J Appl Physiol 23: 331-335, 1967. 45. Fritzsche RG, Switzer TW, Hodgkinson BJ, Lee SH, Martin JC and Coyle EF. Water and carbohydrate ingestion during prolonged exercise increase maximal neuromuscular power. J Appl Physiol 88: 730-737, 2000. 46. Galbo H, Holst JJ and Christensen NJ. Glucagon and plasma catecholamine responses to graded and prolonged exercise in man. J Appl Physiol 38: 70-76, 1975. 47. Gisolfi CV, Summers RW, Schedl HP and Bleiler TL. Intestinal water absorption from select carbohydrate solutions in humans. J Appl Physiol 73: 2142-2150, 1992. 48. Guezennec CY, Satabin P, Duforez F, Merino D, Peronnet F and Koziet J. Oxidation of corn starch, glucose, and fructose ingested before exercise. Med Sci Sports Exerc 21: 45-50, 1989. 97 49. Hargreaves M, Costill DL, Coggan A, Fink WJ and Nishibata I. Effect of carbohydrate feedings on muscle glycogen utilization and exercise performance. Med Sci Sports Exerc 16: 219-222, 1984. 50. Harvey CR, Frew R, Massicotte D, Peronnet F and Rehrer NJ. Muscle glycogen oxidation during prolonged exercise measured with oral [13C]glucose: comparison with changes in muscle glycogen content. J Appl Physiol 102: 1773- 1779, 2007. 51. Hawley JA, Dennis SC and Noakes TD. Oxidation of carbohydrate ingested during prolonged endurance exercise. Sports Med 14: 27-42, 1992. 52. Hawley JA, Dennis SC, Nowitz A, Brouns F and Noakes TD. Exogenous carbohydrate oxidation from maltose and glucose ingested during prolonged exercise. Eur J Appl Physiol Occup Physiol 64: 523-527, 1992. 53. Hermansen L, Hultman E and Saltin B. Muscle glycogen during prolonged severe exercise. Acta Physiol Scand 71: 129-139, 1967. 54. Hermansen L and Vaage O. Lactate disappearance and glycogen synthesis in human muscle after maximal exercise. Am J Physiol 233: E422-E429, 1977. 55. Horowitz JF, Mora-Rodriguez R, Byerley LO and Coyle EF. Substrate metabolism when subjects are fed carbohydrate during exercise. Am J Physiol 276: E828-E835, 1999. 98 56. Ivy JL, Costill DL, Fink WJ and Lower RW. Influence of caffeine and carbohydrate feedings on endurance performance. Med Sci Sports 11: 6-11, 1979. 57. Ivy JL, Miller W, Dover V, Goodyear LG, Sherman WM, Farrell S and Williams H. Endurance improved by ingestion of a glucose polymer supplement. Med Sci Sports Exerc 15: 466-471, 1983. 58. Jandrain B, Krzentowski G, Pirnay F, Mosora F, Lacroix M, Luyckx A and Lefebvre P. Metabolic availability of glucose ingested 3 h before prolonged exercise in humans. J Appl Physiol 56: 1314-1319, 1984. 59. Jentjens RL, Achten J and Jeukendrup AE. High oxidation rates from combined carbohydrates ingested during exercise. Med Sci Sports Exerc 36: 1551-1558, 2004. 60. Jentjens RL and Jeukendrup AE. High rates of exogenous carbohydrate oxidation from a mixture of glucose and fructose ingested during prolonged cycling exercise. Br J Nutr 93: 485-492, 2005. 61. Jentjens RL, Moseley L, Waring RH, Harding LK and Jeukendrup AE. Oxidation of combined ingestion of glucose and fructose during exercise. J Appl Physiol 96: 1277-1284, 2004. 62. Jentjens RL, Shaw C, Birtles T, Waring RH, Harding LK and Jeukendrup AE. Oxidation of combined ingestion of glucose and sucrose during exercise. Metabolism 54: 610-618, 2005. 99 63. Jentjens RL, Underwood K, Achten J, Currell K, Mann CH and Jeukendrup AE. Exogenous carbohydrate oxidation rates are elevated after combined ingestion of glucose and fructose during exercise in the heat. J Appl Physiol 100: 807-816, 2006. 64. Jentjens RL, Venables MC and Jeukendrup AE. Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise. J Appl Physiol 96: 1285- 1291, 2004. 65. Jeukendrup A, Brouns F, Wagenmakers AJ and Saris WH. Carbohydrate- electrolyte feedings improve 1 h time trial cycling performance. Int J Sports Med 18: 125-129, 1997. 66. Jeukendrup AE. Carbohydrate intake during exercise and performance. Nutrition 20: 669-677, 2004. 67. Jeukendrup AE, Borghouts LB, Saris WH and Wagenmakers AJ. Reduced oxidation rates of ingested glucose during prolonged exercise with low endogenous CHO availability. J Appl Physiol 81: 1952-1957, 1996. 68. Jeukendrup AE, Mensink M, Saris WH and Wagenmakers AJ. Exogenous glucose oxidation during exercise in endurance-trained and untrained subjects. J Appl Physiol 82: 835-840, 1997. 100 69. Jeukendrup AE, Moseley L, Mainwaring GI, Samuels S, Perry S and Mann CH. Exogenous carbohydrate oxidation during ultraendurance exercise. J Appl Physiol 100: 1134-1141, 2006. 70. Jeukendrup AE, Wagenmakers AJ, Stegen JH, Gijsen AP, Brouns F and Saris WH. Carbohydrate ingestion can completely suppress endogenous glucose production during exercise. Am J Physiol 276: E672-E683, 1999. 71. Jeukendrup AE and Wallis GA. Measurement of substrate oxidation during exercise by means of gas exchange measurements. Int J Sports Med 26 Suppl 1: S28-S37, 2005. 72. Karlsson J and Saltin B. Diet, muscle glycogen, and endurance performance. J Appl Physiol 31: 203-206, 1971. 73. Koivisto VA, Harkonen M, Karonen SL, Groop PH, Elovainio R, Ferrannini E, Sacca L and Defronzo RA. Glycogen depletion during prolonged exercise: influence of glucose, fructose, or placebo. J Appl Physiol 58: 731-737, 1985. 74. Krzentowski G, Pirnay F, Luyckx AS, Lacroix M, Mosora F and Lefebvre PJ. Effect of physical training on utilization of a glucose load given orally during exercise. Am J Physiol 246: E412-E417, 1984. 75. Kuipers H, Costill DL, Porter DA, Fink WJ and Morse WM. Glucose feeding and exercise in trained rats: mechanisms for glycogen sparing. J Appl Physiol 61: 859- 863, 1986. 101 76. Leese GP, Nicoll AE, Varnier M, Thompson J, Scrimgeour CM and Rennie MJ. Kinetics of 13CO2 elimination after ingestion of 13C bicarbonate: the effects of exercise and acid base balance. Eur J Clin Invest 24: 818-823, 1994. 77. Madsen K, MacLean DA, Kiens B and Christensen D. Effects of glucose, glucose plus branched-chain amino acids, or placebo on bike performance over 100 km. J Appl Physiol 81: 2644-2650, 1996. 78. Massicotte D, Hillaire-Marcel C, Ledoux M and Peronnet F. The natural isotope tracing with 13C: a non-invasive method for studying the metabolism during exercise. Can J Appl Sport Sci 9: 164, 1984. 79. Massicotte D, Peronnet F, Adopo E, Brisson GR and Hillaire-Marcel C. Metabolic availability of oral glucose during exercise: a reassessment. Metabolism 41: 1284-1290, 1992. 80. Massicotte D, Peronnet F, Allah C, Hillaire-Marcel C, Ledoux M and Brisson G. Metabolic response to [13C]glucose and [13C]fructose ingestion during exercise. J Appl Physiol 61: 1180-1184, 1986. 81. Massicotte D, Peronnet F, Brisson G, Bakkouch K and Hillaire-Marcel C. Oxidation of a glucose polymer during exercise: comparison with glucose and fructose. J Appl Physiol 66: 179-183, 1989. 102 82. Massicotte D, Peronnet F, Pitre C, Adopo E, Brisson GR and Hillaire-Marcel C. Exogenous 13C glucose oxidation during exercise: North American vs Western European studies. Eur J Appl Physiol Occup Physiol 67: 402-407, 1993. 83. Maughan RJ, Bethell LR and Leiper JB. Effects of ingested fluids on exercise capacity and on cardiovascular and metabolic responses to prolonged exercise in man. Exp Physiol 81: 847-859, 1996. 84. Maughan RJ, Fenn CE and Leiper JB. Effects of fluid, electrolyte and substrate ingestion on endurance capacity. Eur J Appl Physiol Occup Physiol 58: 481-486, 1989. 85. McConell G, Fabris S, Proietto J and Hargreaves M. Effect of carbohydrate ingestion on glucose kinetics during exercise. J Appl Physiol 77: 1537-1541, 1994. 86. McHugh PR and Moran TH. Calories and gastric emptying: a regulatory capacity with implications for feeding. Am J Physiol 236: R254-R260, 1979. 87. Millard-Stafford M, Sparling PB, Rosskopf LB, Hinson BT and DiCarlo LJ. Carbohydrate-electrolyte replacement during a simulated triathlon in the heat. Med Sci Sports Exerc 22: 621-628, 1990. 88. Mitchell JB, Braun WA, Pizza FX and Forrest M. Pre-exercise carbohydrate and fluid ingestion: influence of glycemic response on 10-km treadmill running performance in the heat. J Sports Med Phys Fitness 40: 41-50, 2000. 103 89. Mitchell JB, Costill DL, Houmard JA, Fink WJ, Pascoe DD and Pearson DR. Influence of carbohydrate dosage on exercise performance and glycogen metabolism. J Appl Physiol 67: 1843-1849, 1989. 90. Mitchell JB, Costill DL, Houmard JA, Flynn MG, Fink WJ and Beltz JD. Effects of carbohydrate ingestion on gastric emptying and exercise performance. Med Sci Sports Exerc 20: 110-115, 1988. 91. Moran TH and McHugh PR. Distinctions among three sugars in their effects on gastric emptying and satiety. Am J Physiol 241: R25-R30, 1981. 92. Murray R, Eddy DE, Murray TW, Seifert JG, Paul GL and Halaby GA. The effect of fluid and carbohydrate feedings during intermittent cycling exercise. Med Sci Sports Exerc 19: 597-604, 1987. 93. Murray R, Paul GL, Seifert JG and Eddy DE. Responses to varying rates of carbohydrate ingestion during exercise. Med Sci Sports Exerc 23: 713-718, 1991. 94. Murray R, Paul GL, Seifert JG, Eddy DE and Halaby GA. The effects of glucose, fructose, and sucrose ingestion during exercise. Med Sci Sports Exerc 21: 275- 282, 1989. 95. Murray R, Seifert JG, Eddy DE, Paul GL and Halaby GA. Carbohydrate feeding and exercise: effect of beverage carbohydrate content. Eur J Appl Physiol Occup Physiol 59: 152-158, 1989. 104 96. Neufer PD, Costill DL, Fink WJ, Kirwan JP, Fielding RA and Flynn MG. Effects of exercise and carbohydrate composition on gastric emptying. Med Sci Sports Exerc 18: 658-662, 1986. 97. Neufer PD, Costill DL, Flynn MG, Kirwan JP, Mitchell JB and Houmard J. Improvements in exercise performance: effects of carbohydrate feedings and diet. J Appl Physiol 62: 983-988, 1987. 98. Nicholas CW, Tsintzas K, Boobis L and Williams C. Carbohydrate-electrolyte ingestion during intermittent high-intensity running. Med Sci Sports Exerc 31: 1280-1286, 1999. 99. Nicholas CW, Williams C, Lakomy HK, Phillips G and Nowitz A. Influence of ingesting a carbohydrate-electrolyte solution on endurance capacity during intermittent, high-intensity shuttle running. J Sports Sci 13: 283-290, 1995. 100. Noakes TD, Lambert EV, Lambert MI, McArthur PS, Myburgh KH and Benade AJ. Carbohydrate ingestion and muscle glycogen depletion during marathon and ultramarathon racing. Eur J Appl Physiol Occup Physiol 57: 482-489, 1988. 101. Osterberg KL, Zachwieja JJ and Smith JW. Carbohydrate and carbohydrate + protein for cycling time-trial performance. J Sports Sci 26: 227-233, 2008. 102. Pallikarakis N, Jandrain B, Pirnay F, Mosora F, Lacroix M, Luyckx AS and Lefebvre PJ. Remarkable metabolic availability of oral glucose during long- duration exercise in humans. J Appl Physiol 60: 1035-1042, 1986. 105 103. Pallikarakis N, Sphiris N and Lefebvre P. Influence of the bicarbonate pool and on the occurrence of 13CO2 in exhaled air. Eur J Appl Physiol Occup Physiol 63: 179-183, 1991. 104. Peronnet F, Adopo E, Massicotte D and Hillaire-Marcel C. Exogenous substrate oxidation during exercise: studies using isotopic labelling. Int J Sports Med 13 Suppl 1: S123-S125, 1992. 105. Peronnet F and Massicotte D. Table of nonprotein respiratory quotient: an update. Can J Sport Sci 16: 23-29, 1991. 106. Peronnet F, Massicotte D, Brisson G and Hillaire-Marcel C. Use of 13C substrates for metabolic studies in exercise: methodological considerations. J Appl Physiol 69: 1047-1052, 1990. 107. Peronnet F, Massicotte D, Folch N, Melin B, Koulmann N, Jimenez C, Bourdon L, Launay JC and Savourey G. Substrate utilization during prolonged exercise with ingestion of (13)C-glucose in acute hypobaric hypoxia (4,300 m). Eur J Appl Physiol 97: 527-534, 2006. 108. Peronnet F, Rheaume N, Lavoie C, Hillaire-Marcel C and Massicotte D. Oral [13C]glucose oxidation during prolonged exercise after high- and low- carbohydrate diets. J Appl Physiol 85: 723-730, 1998. 106 109. Pirnay F, Crielaard JM, Pallikarakis N, Lacroix M, Mosora F, Krzentowski G, Luyckx AS and Lefebvre PJ. Fate of exogenous glucose during exercise of different intensities in humans. J Appl Physiol 53: 1620-1624, 1982. 110. Pirnay F, Lacroix M, Mosora F, Luyckx A and Lefebvre P. Glucose oxidation during prolonged exercise evaluated with naturally labeled [13C]glucose. J Appl Physiol 43: 258-261, 1977. 111. Radziuk J and Bondy DC. Abnormal oral glucose tolerance and glucose malabsorption after vagotomy and pyloroplasty. A tracer method for measuring glucose absorption rates. Gastroenterology 83: 1017-1025, 1982. 112. Ravich WJ, Bayless TM and Thomas M. Fructose: incomplete intestinal absorption in humans. Gastroenterology 84: 26-29, 1983. 113. Ravussin E, Pahud P, Dorner A, Arnaud MJ and Jequier E. Substrate utilization during prolonged exercise preceded by ingestion of 13C-glucose in glycogen depleted and control subjects. Pflugers Arch 382: 197-202, 1979. 114. Rehrer NJ, Wagenmakers AJ, Beckers EJ, Halliday D, Leiper JB, Brouns F, Maughan RJ, Westerterp K and Saris WH. Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise. J Appl Physiol 72: 468-475, 1992. 107 115. Riddell MC, Bar-Or O, Schwarcz HP and Heigenhauser GJ. Substrate utilization in boys during exercise with [13C]-glucose ingestion. Eur J Appl Physiol 83: 441- 448, 2000. 116. Rogers J, Summers RW and Lambert GP. Gastric emptying and intestinal absorption of a low-carbohydrate sport drink during exercise. Int J Sport Nutr Exerc Metab 15: 220-235, 2005. 117. Rumessen JJ and Gudmand-Hoyer E. Absorption capacity of fructose in healthy adults. Comparison with sucrose and its constituent monosaccharides. Gut 27: 1161-1168, 1986. 118. Ruzzin J, Peronnet F, Tremblay J, Massicotte D and Lavoie C. Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose. J Appl Physiol 95: 477-482, 2003. 119. Ryan AJ, Lambert GP, Shi X, Chang RT, Summers RW and Gisolfi CV. Effect of hypohydration on gastric emptying and intestinal absorption during exercise. J Appl Physiol 84: 1581-1588, 1998. 120. Saris WH, Goodpaster BH, Jeukendrup AE, Brouns F, Halliday D and Wagenmakers AJ. Exogenous carbohydrate oxidation from different carbohydrate sources during exercise. J Appl Physiol 75: 2168-2172, 1993. 108 121. Sasaki H, Maeda J, Usui S and Ishiko T. Effect of sucrose and caffeine ingestion on performance of prolonged strenuous running. Int J Sports Med 8: 261-265, 1987. 122. Satabin P, Portero P, Defer G, Bricout J and Guezennec CY. Metabolic and hormonal responses to lipid and carbohydrate diets during exercise in man. Med Sci Sports Exerc 19: 218-223, 1987. 123. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ and Stachenfeld NS. American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports Exerc 39: 377-390, 2007. 124. Simard C, Tremblay A and Jobin M. Effects of carbohydrate intake before and during an ice hockey game on blood and muscle energy substrates. Research Quarterly for Exercise and Sport 59: 144-147, 1988. 125. Slentz CA, Davis JM, Settles DL, Pate RR and Settles SJ. Glucose feedings and exercise in rats: glycogen use, hormone responses, and performance. J Appl Physiol 69: 989-994, 1990. 126. Sonne B and Galbo H. Carbohydrate metabolism in fructose-fed and food- restricted running rats. J Appl Physiol 61: 1457-1466, 1986. 127. Sugiura K and Kobayashi K. Effect of carbohydrate ingestion on sprint performance following continuous and intermittent exercise. Med Sci Sports Exerc 30: 1624-1630, 1998. 109 128. Trimmer JK, Casazza GA, Horning MA and Brooks GA. Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions. Am J Physiol Endocrinol Metab 281: E683-E692, 2001. 129. Tsintzas K and Williams C. Human muscle glycogen metabolism during exercise. Effect of carbohydrate supplementation. Sports Med 25: 7-23, 1998. 130. Tsintzas OK, Williams C, Boobis L and Greenhaff P. Carbohydrate ingestion and single muscle fiber glycogen metabolism during prolonged running in men. J Appl Physiol 81: 801-809, 1996. 131. Tsintzas OK, Williams C, Boobis L and Greenhaff P. Carbohydrate ingestion and glycogen utilization in different muscle fibre types in man. J Physiol 489 ( Pt 1): 243-250, 1995. 132. Tsintzas OK, Williams C, Wilson W and Burrin J. Influence of carbohydrate supplementation early in exercise on endurance running capacity. Med Sci Sports Exerc 28: 1373-1379, 1996. 133. Van Essen M. and Gibala MJ. Failure of protein to improve time trial performance when added to a sports drink. Med Sci Sports Exerc 38: 1476-1483, 2006. 134. Vergauwen L, Brouns F and Hespel P. Carbohydrate supplementation improves stroke performance in tennis. Med Sci Sports Exerc 30: 1289-1295, 1998. 110 135. Vist GE and Maughan RJ. Gastric emptying of ingested solutions in man: effect of beverage glucose concentration. Med Sci Sports Exerc 26: 1269-1273, 1994. 136. Wagenmakers AJ, Brouns F, Saris WH and Halliday D. Oxidation rates of orally ingested carbohydrates during prolonged exercise in men. J Appl Physiol 75: 2774-2780, 1993. 137. Wallis GA, Yeo SE, Blannin AK and Jeukendrup AE. Dose-response effects of ingested carbohydrate on exercise metabolism in women. Med Sci Sports Exerc 39: 131-138, 2007. 138. Williams C, Nute MG, Broadbank L and Vinall S. Influence of fluid intake on endurance running performance. Eur J Appl Physiol 60: 112-119, 1990. 139. Winnick JJ, Davis JM, Welsh RS, Carmichael MD, Murphy EA and Blackmon JA. Carbohydrate feedings during team sport exercise preserve physical and CNS function. Med Sci Sports Exerc 37: 306-315, 2005. 140. Wolfe RR, Nadel ER, Shaw JH, Stephenson LA and Wolfe MH. Role of changes in insulin and glucagon in glucose homeostasis in exercise. J Clin Invest 77: 900- 907, 1986. 141. Wolfe RR, Shaw JH, Nadel ER and Wolfe MH. Effect of substrate intake and physiological state on background 13CO2 enrichment. J Appl Physiol 56: 230- 234, 1984. 111 142. Wright DA, Sherman WM and Dernbach AR. Carbohydrate feedings before, during, or in combination improve cycling endurance performance. J Appl Physiol 71: 1082-1088, 1991. 143. Yaspelkis BB, III, Patterson JG, Anderla PA, Ding Z and Ivy JL. Carbohydrate supplementation spares muscle glycogen during variable-intensity exercise. J Appl Physiol 75: 1477-1485, 1993. 144. Zanconato S, Cooper DM, Barstow TJ and Landaw E. 13CO2 washout dynamics during intermittent exercise in children and adults. J Appl Physiol 73: 2476-2482, 1992. 112 APPENDICES 113 APPENDIX A: INFORMED CONSENT GATORADE SPORTS SCIENCE INSTITUTE SUBJECT INFORMED CONSENT ? PHYSIOLOGY RESEARCH PROJECT LIGHT WEIGHT: Two hour fixed-load cycling followed by a simulated 20- kilometer time trial. RESEARCHERS: JohnEric Smith, Jeff Zachwieja Please read the following information carefully and feel free to ask questions. Sign the final page only when you are satisfied that all procedures and risks have been sufficiently explained to you. A. REQUIREMENTS This study requires that you meet the following criteria: ? You must have undergone stress-testing and be cleared for participation by the medical director of GSSI. ? You must be a non-smoking male between the ages of 18-40 with no history of diabetes or PKU (phenylketonuria). You must be used to cycling for long periods of time at a moderate to high intensity. 114 Subjects may be excluded following one or more of the pre-tests based on fitness level or inability to complete the protocol. B. PURPOSE OF THE STUDY This study is designed to determine the effect of drinking sports drinks with different nutrient compositions on cycling performance. C. TEST PROCEDURES On nine (9) separate occasions, you will be asked to ride your own bike on a CompuTrainer in our laboratory. NINE VISITS TO THE LAB WILL BE REQUIRED, AS SUMMARIZED BELOW: 1. VO 2 Max Test: This test will involve cycling for 2-3 minute periods at an increasingly higher resistance until you can no longer pedal. We will measure your peak oxygen consumption rate and peak cycling wattage. To assess oxygen consumption rate, you will be required to perform the test with a mask placed over your mouth and nose. You will be able to easily inhale room air while your expired air will be analyzed for oxygen and carbon dioxide content (Total time: 45 minutes). 2. Lactate Threshold Test: This test will consist of riding for 3 minute stages at 50, 55, 60, 65, 70, 75, 80, 85, and 90 percent of your VO 2 max (determined in the test above). Blood samples will be taken to measure the amount of lactate in your blood. A registered Nurse will insert a small sterile flexible tube into one of the arm veins that is close to the skin to get blood samples. The sterile tube will remain in your arm vein 115 for the length of the experiment. Approximately 4 ml of blood will be drawn at each stage of the test totaling about 30 ml (1 oz) of blood for the entire test. All equipment used in the procedure is sterile. You will wear a mask for this test in order to assess your oxygen consumption as in the VO 2 Max Test. (Total time: 60 minutes) 3. Familiarization I: The purpose of this visit is to allow you to become familiar with the CompuTrainer 20 kilometer time trial course. (Total time 1 hour) 4. Familiarization II: The purpose of this visit is to allow you to become familiar with riding the CompuTrainer 20 kilometer time trial course after a two hour constant resistance ride that will be set just below your lactate threshold. (Total time: 3 hours) 5. Familiarization III: The purpose of this visit is for you to become familiar with the drinking schedule and physiological measures that will be made during the experiment. During the 2 hour constant resistance rise (resistance set just below your lactate threshold) you will receive about 8.5 oz of water every 15 minutes. A registered Nurse will insert a small sterile flexible tube into one of the arm veins that is close to the skin to get blood samples. The sterile tube will remain in your arm vein for the length of the experiment. Approximately 8 ml of blood will be drawn before the initiation of the constant resistance ride and then every 15 minutes during the last hour of the constant resistance ride totaling 48 ml (~1.6 oz) of blood for the entire test. All equipment used in the procedure is sterile. Heart rate and oxygen consumption will be monitored and ratings of perceived exertion will be recorded throughout. When the 2-hour constant resistance ride is complete, you will receive a final 8.5 oz of water and a two minute rest. You will then complete a simulated 20-kilometer time 116 trial. The goal of the time trial will be to complete the distance as quickly as you can. Heart rate will be monitored throughout the time trial. (Total time: 4 hours) 6. Experimental treatment I - IV: Performance trials with test beverages. The 2 hour constant resistance ride will be set just below your lactate threshold. You will receive about 8.5 oz of a beverage every 15 minutes during the constant resistance ride. A registered Nurse will insert a small sterile flexible tube into one of the arm veins that is close to the skin to get blood samples. The sterile tube will remain in your arm vein for the length of the experiment. Approximately 8 ml of blood will be drawn before the initiation of the constant resistance ride and then every 15 minutes during the last hour of the constant resistance ride totaling 48 ml (~1.6 oz) of blood for the entire test. All equipment used in the procedure is sterile. Heart rate and oxygen consumption will be monitored and ratings of perceived exertion will be recorded throughout. When the 2-hour constant resistance ride is complete, you will receive a final 8.5 oz of beverage and a two minute rest. You will then complete a simulated 20-kilometer time trial. The goal of the time trial will be to complete the distance as quickly as you can. Heart rate will be monitored throughout the time trial. (Total time: 4 hours) EXPERIMENTAL SESSIONS: Each session will require the following: ? You MUST arrive 15 minutes prior to the start time (5:45 am). ? You will be asked to fast overnight leading up to the day of an experimental trial. On the morning of an experimental trial you are permitted water, but no other food or beverage. 117 ? No exercise for 24 hours prior to the testing. ? You will be asked to keep a written food record of your diet for 24- hours prior to the first experimental trial. You will repeat that diet for each of the 3 subsequent experiments. ? You will be required to empty your bladder just prior to each experiment. If you need to urinate during a test you will collect it in a plastic container, which will be weighed and then disposed. ? Nude bodyweights will be measured before and after the experiment. This occurs in privacy behind a screened off area. Your weight is recorded from a digital readout outside of the privacy area. ? You will be asked to consume each of the beverages given to you in their entirety during exercise. D. HEALTH RISKS All experimental procedures used in this study have been routinely used in this and other exercise physiology laboratories, and present minimal risk to your health. However, you should be aware that there are risks involved in any laboratory procedure. ? Exercise: Abnormal responses to exercise include unusual blood pressure responses, disturbances in heart function, nausea, and fainting. Risks also include muscle cramps, strains, tears, joint and/or muscle pain, sweating, breathlessness, breathing difficulty, changes in heart rate, stroke and death. If any of these symptoms occur during the exercise session, stop exercising and notify a staff 118 member immediately. In addition a fall from the bike may result in bruises, broken bones, dislocations, or head injury. ? Dehydration: Risks associated with dehydration include abnormal feelings of fatigue, irritability, headache, lightheadedness, abnormal heart rate and blood pressure response and heat illness. Despite drinking fluids during exercise modest dehydration may occur. This level of dehydration (< 1%) is commonly experienced by athletes with no associated symptoms. However, every precaution, including provision of beverage will be taken to reduce all risks of dehydration. ? Blood Draw: Pain or infection may occur. ? Beverage Ingredients: The beverages used in this study will contain electrolytes and various levels of glucose. Glucose (C 6 H 12 O 6 ) is made up of carbon (C), hydrogen (H), and oxygen (O). Most of the carbon in nature has a mass of 12, a small portion of the glucose you will ingest will be enriched with carbon 13 ( 13 C), a naturally occurring isotope of carbon that is non-radioactive. The 13 C will allow us to monitor how much of the ingested glucose is being used by the muscle during the exercise. Some beverages contain FDA approved artificial sweeteners, which are harmful to individuals who have the metabolic condition PKU. E. NOTIFICATION TO STAFF For your health and safety, it is imperative that you notify the lab staff of any acute and/or chronic medical conditions, and your current use of all medications (even over- the-counter drugs). You are also required to notify us of any allergies you may have. 119 It is also important that you immediately notify the lab staff during exercise of any unusual symptoms or abnormal responses arising from the testing. Emergency procedures are coordinated through the Barrington Paramedics and Good Shepherd Hospital. All precautions needed to minimize risks to your health will be taken. F. ACCESS TO TEST RESULTS Upon specific request, you may have access to certain test results, provided those results do not reveal any information that is confidential to GSSI research. G. CONFIDENTIALITY OF TEST RESULTS Your test results will be kept in the Gatorade Sports Science Institute laboratories. Grouped or blind-coded data may be used for publication in scientific journals or for marketing claims. Personal medical confidentiality will not be breached. Only the staff will have access to your individual test results. H. PUBLICITY RELEASE As part of my participation in testing by the Gatorade Sport Science Institute, I understand that photographs, videotapes and/or drawings or other likenesses of me may be taken and used from time to time by the press or by Gatorade for public relations or other publicity or advertising purposes. I hereby grant full permission to Stokely-Van Camp, Inc. and The Quaker Oats Company, and their parents, affiliates, successors and assigns or anyone authorized by any of them, to use my name, photograph, video image, voice, likeness and biographical data, in whole or in part, in any and all media for the 120 purposes of publicity, advertising, trade or news purposes and in connection therewith I hereby release them and each of them from all liability. I have read each and every word of the foregoing prior to my execution of this release and am fully familiar with the contents thereof. I. SUBJECT COMPENSATION After all testing has been completed, you will receive an honorarium that includes: $ 25 for VO 2 max test $ 25 for lactate threshold test $ 25 for familiarization I trial $ 75 for familiarization II trial $100 for familiarization III trial $100 for each of 4 experimental sessions (4 tests X $100 = $400) $ 50 finishing bonus Total compensation upon completion of all tests = $700 You may withdraw at any time from any or all sessions without prejudice to your status at PepsiCo, or as a subject in the GSSI research program. If you withdraw, your honorarium will be prorated based on the number of sessions completed. The honorarium will also be prorated if a test is partially completed and must be rescheduled due to staff constraints or experimental failure of our equipment. 121 J. INFORMED CONSENT FORM AND WAIVER FORM By reading and signing this document, I acknowledge my consent to participate based on sections ?A? through ?I? above. I also acknowledge that my participation in this exercise activity is not without risk of injury. I understand that muscular or orthopedic injury may result from my improper use of the equipment, from poor exercise technique, and from overuse or overtraining. Although I have been screened by GSSI staff or physician, including a symptom-limited graded exercise test, there exists the possibility of certain changes occurring during exercise. These include abnormal blood pressure, fainting, irregular, fast or slow heart rhythm, and in rare instances, heart attack, stroke or death. I am aware that every effort will be made to minimize these risks by provision of appropriate supervision during exercise. Emergency equipment and trained personnel are available to deal with unusual situations that may arise. I declare that I am in good physical and mental health, and have no heart, lung, kidney, musculoskeletal disease or problems, or diabetes mellitus or any disorder that would make my participation medically inadvisable. I hereby release from liability and promise not to sue Pepsico, Inc, Stokely Van-Camp, and the Gatorade Company, and any of their subsidiaries, affiliates, officers and employees, for all loss or damage suffered by me or to my bicycle, and I promise not to make any claim on account of injury to my person or property or resulting in my death whether caused by negligence or otherwise to myself, my personal representatives, heirs and next of kin. I have been given ample opportunity to read this document and to ask questions which have been answered to my satisfaction. I understand the intent of this document 122 and acknowledge the possibility of exercise-related injury. I hereby consent to participate in this exercise activity, under the terms and conditions stated above. I know that I may withdraw my consent and stop participation in the exercise session at anytime, for any reason. I agree to the terms set forth in the subject informed consent document and further agree to keep confidential any information learned or obtained in connection with my participation in the exercise activity and to not disclose to others my impression of or use in any way any information that is confidential or proprietary to PepsiCo, Inc concerning any concepts, proposals, test results, improvements to existing products or any other confidential or proprietary information. I sign this document voluntarily. Signature ____________________________________ Date:______________ Name (Printed) ________________________________ Signature of Investigator: ________________________ Date:______________ 123 APPENDIX B: DATA COLLECTION SHEET Participant _______________ Trial Number ____________ Date ______________ o Get dietary record from participant. o Have the participant empty their bladder record a nude body weight Body Weight ____________ kg Urine Volume _____________ g Urine Specific Gravity ______________ o Have nurse insert catheter o Collect 1 red top vacutainer of blood 2 green top vacutainers of blood 1 hepranized syringe of blood After Catheter Insertion o Collect and analyze one 2 minute Douglas Bag sample. O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ o Set the bike up on the trainer. o Remove participant?s cyclocomputer. o Remove participant?s watch. o Have participant put on Polar Heart Rate monitor. o Change the rider to the participant?s name. o Provide the participant with a towel. 124 o Have the participant ride 10 minutes at 100 Watts for a warm-up. o Following the warm-up have the participant calibrate the trainer. Set the number between 2.0 and 2.1 ____________ o Have the participant mount the bike and begin the 2 hour ride at the predetermined workload Workload _______________ Watts. Gearing: Front _____________ Rear ______________ o Turn front fan on 1. At 13 minutes o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Give 1 st Beverage At 28 minutes 125 o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Give 2 nd Beverage At 43 minutes o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Give 3 rd Beverage At 58 minutes 126 o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Collect 1 red top vacutainer of blood 2 green top vacutainers of blood 1 hepranized syringe of blood o Give 4 th Beverage At 73 minutes o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ 127 Barometric Pressure __________ o Collect 1 red top vacutainer of blood 2 green top vacutainers of blood 1 hepranized syringe of blood o Give 5 th Beverage At 88 minutes o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Collect 1 red top vacutainer of blood 2 green top vacutainers of blood 1 hepranized syringe of blood o Give 6 th Beverage At 103 minutes o Record RPE rating ______________ o Record Heart Rate ______________ 128 o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Collect 1 red top vacutainer of blood 2 green top vacutainers of blood 1 hepranized syringe of blood o Give 7 th Beverage At 118 minutes o Record RPE rating ______________ o Record Heart Rate ______________ o Collect and analyze two 30 second Douglas Bag sample. . O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ O 2 % __________ CO 2 % __________ Sampling Time ________ Initial Volumetric Reading ________ Final Volumetric Reading ________ Room Temperature __________ Room Humidity __________ Barometric Pressure __________ o Collect 1 red top vacutainer of blood 129 2 green top vacutainers of blood 1 hepranized syringe of blood o Give 8 th Beverage At 120 minutes o Allow the participant to dismount the bike o Turn off any music o Lock doors o Select 20 k time trial 3 and desert view At 121:30 o Have participant mount bike o Have participant finish beverage At 121:57 o Press start to begin time trial o Record completion time : : . hrs mins sec hundredths o Record participant?s nude body weight Body Weight ____________ kg o Have the participant empty their bladder into a cup and record urine volume Urine Volume ____________ g Urine Specific Gravity ___________ o Allow the participant to cool down and provide with their choice of beverage. 130 Research Initials: 1. __________ 2. __________ 3. __________