Oxidative Stres and Heart Rate Variability Following an Acute Bout of CrossFit by Brian Mathew Kliszczewicz A disertation submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 4, 2014 Keywords: Oxidative-stres, Heart Rate Variability, CrossFit, High-intensity Copyright 2014 by Brian Mathew Kliszczewicz Approved by Daniel Blesing, Chair, Profesor of Kinesiology John Quindry, Asociate Profesor of Kinesiology Gretchen Oliver, Asistant Profesor of Kinesiology Michael Esco, Asociate Profesor of Physical Education and Exercise Science ii Abstract The purpose of this Disertation was to provide a beter understanding of the physiological stres involved in the newly popular style of exercise named CrossFit, and compare these findings to a more ?traditional? mode of exercise, treadmil running. Heart Rate Variability (HRV), which is a marker of parasympathetic activity and cardiac autonomic control, and oxidative stres, which are biomarkers of celular damage are both independent markers of physiological stres. This disertation covers two studies. The first study examined the afects of CrossFit and an intensity-matched bout of treadmil running on markers of HRV in ten CrossFit trained participants. The results of this study showed that both markers of HRV (LnRMSD, LnHF) significantly decreased following both CrossFit and treadmil running. Furthermore, both markers of HRV were significantly lower following the trial when compared to treadmil running. The second study examined biomarkers of oxidative-stres following a bout of CrossFit and intensity-matched bout of treadmil running. Ten CrossFit Trained male athletes participated in this study. Markers of oxidative stres can be divided into two categories: oxidative-damage (LOH, PC), and antioxidant capacity (FRAP, TEAC). The results of this study were mixed, with biomarkers of oxidative-stres LOH and FRAP becoming significantly elevated following both trials and an unexpected significant decrease in PC and TEAC. No biomarker showed a trial dependent diference. iii Acknowledgments I would like to thank Dr. Daniel Blesing for agreeing to be my commite chair and advisor during my pursuit for my Doctorate of Philosophy in Exercise Science and my career in Exercise Physiology. I also would like to thank the members of my commite: Dr. John Quindry, Dr. Gretchen Oliver, and Dr. Michael Esco for their enormous contribution to this project. I would like to give special thanks Dr. Michael Esco and Dr. John Quindry for the generous use of their facilities and supplies, for without them this project would not have been possible. I would also like to thank Dr. Barbara Wilder for agreeing to be my outside reader. Furthermore, I would like to expres my gratitude to the Profesors of Auburn University, whose influence brought me to this acomplishment. In addition, I would like to thank Dr. Kyle Taylor for his enormous commitment to this project, his wilingnes and generosity alowed for the completion of this study. I would also like to thank Dr. J.C. Bradbury for giving me the opportunity to begin my career as an Asistant Profesor at Kennesaw State University. To my friends and family, I would like to expres my deepest thanks. The patients and support given to me through this proces was nothing short of amazing. None of this would have even been possible, let alone finished without the incredible influences form you al. From the bottom of my heart I thank you al. iv Table of Contents Abstract.........................................................................................................................................ii Acknowledgments.......................................................................................................................iii List of Tables................................................................................................................................v List of Figures..............................................................................................................................vi List of Appendices......................................................................................................................vi List of Abbreviations.................................................................................................................vii Chapter 1 INTRODUCTION.......................................................................................................1 Significance of Study ......................................................................................................3 Statement of Research Objectives ..................................................................................5 Chapter 2 LITERATURE REVIEW ...........................................................................................6 References .................................................................................................................................30 Chapter 3 ACUTE EXERCISE AND HEART RATE VARIABILITY: CROSFIT VS. CONTINUOUS RUNING ......................................................................................................38 References .................................................................................................................................52 Chapter 4 ACUTE EXERCISE AND OXIDATIVE STRES: CROSFIT VS. TREADMILL BOUT..........................................................................................................................................60 References .................................................................................................................................76 v List of Tables Chapter 2 Table 1. Example CrossFit Workouts ........................................................................................37 Chapter 3 Table 1 Participant Characteristics ...........................................................................................55 Table 2 Exercise Intensity ..........................................................................................................56 Chapter 4 Table 1 Participant Characteristics.............................................................................................79 Table 2 Exercise Intensity ..........................................................................................................78 vi List of Figures Chapter 3 Figure 1 Study Design ...............................................................................................................57 Figure 2 Pre and Post markers of HRV......................................................................................58 Chapter 4 Figure 1 Study Design ...............................................................................................................81 Figure 2 Pre and Post markers of Oxidative Damage.................................................................82 Figure 3 Pre and Post markers of Antioxidant Capacity ...........................................................83 vii List of Appendices Chapter 3 Appendix A Raw RMSD & HF ...............................................................................................59 vii List of Abbreviations CF CrossFit ECG Electrocardiogram FRAP Ferric-reducing antioxidant potential HF High frequency domain HIIT High-intensity interval training HIPT High-intensity power training HR Heart rate HR max Heart rate max HRV Heart rate variability LF Low frequency domain LF:HF High frequency to low frequency ratio LnHF Log transformed high frequency domain LnRMSD Log transformed square root of the mean sum of the squares of diferences betwen adjacent R to R (normal to normal) intervals LOOH Lipid hydroperoxides PC Protein Carbonyl RMSD Square root of the mean sum of the squares of diferences betwen adjacent R to R (normal to normal) intervals TEAC Trolox equivalent antioxidant capacity ix TM Treadmil VLF Very low frequency domain ! "! CHAPTER I INTRODUCTION High intensity power training (HIPT) has become a popular form of exercise. The relatively new style of training involves multiple joint resistance exercises while atempting to maintain high power outputs (63). The best-known form of HIPT, CrossFit, is globaly popular and has garnered a strong faction. The growth in popularity has occurred for several reasons: easy aces to programing, a short time commitment, and anecdotal results in body composition and aerobic fitnes. Despite these claims, there is only one scientific study currently available on CrossFit (63). Therefore, it is evident that much of the claims behind the efectivenes of CrossFit are unsubstantiated. Although there is very litle information specificaly about CrossFit within exercise science literature, it is clear that the approach to exercise training is physicaly demanding. Exercise poses a chalenge to the maintenance of homeostasis. However, the disruption of homeostasis becomes greater as the intensity increases (55, 65). Heart rate variability (HRV) and oxidative stres are two independent physiological markers that are often used to evaluate alterations of cardiovascular activity and physiological stres, respectively. Research determining post exercise HRV and oxidative stres following a bout of CrossFit is warranted. The following paragraphs examine these markers as they relate to exercise and risks to health. ! #! Heart rate variability (HRV) is commonly used in research and clinical setings to noninvasively ases autonomic nervous system odulation of the cardiovascular system (20?24, 51?53, 66). HRV involves the measurement of the oscilations that occur betwen consecutive R-R intervals derived from an electrocardiograph (ECG). During exercise a shift of autonomic control from parasympathetic to sympathetic dominance occurs, resulting in acute drop in HRV (7, 11, 31, 49, 50). Depresion of HRV continues for a prolonged period of time following the cesion of an acute bout of exercise. The magnitude and duration of depresion is dependent on the intensity or modality of exercise (31, 62). Post exercise depresion of HRV is asociated with dyshomeostasis and decreases in performance (18, 65). Interestingly, the rate of HRV return miics that of physical recovery (18, 65), which makes HRV an important marker for post high- intensity exercise. Oxidative stres results from free radical damage. Free radicals are atoms or molecules that contain one or more unpaired electrons, which make them highly reactive (4, 19). Oxidative stres is the bi-product of normal celular activity and is commonly referred to in literature as reactive oxygen species (ROS). Increased levels of ROS are central to dyshomeostasis during exercise by means of protein damage and lipid damage (4, 16, 17, 47). Acute exercise leads to increased ROS production and consequent oxidative stres (2, 33, 46, 56, 57). The duration and magnitude of ROS production is proportional to the oxidative stres that is experienced. Low to moderate levels of ROS and subsequent levels of oxidative stres are considered healthy and fundamental to celular adaptive responses to exercise training (4, 43). Although chronicaly elevated levels of ROS can be indicative of pathology (48, 64, 67), the response to acute physical ! $! activity is proportional to the stres experience (2, 3, 8, 34), and therefore indicative of physiological stres. ! %&!'())*+,-!.(/!01!02/!2342!3&0/&'30,!&*0(+/!15!6+1'730!30!3'!+/*'1&*89/!01! /:;/<0!*!4+/*0/+!'2350!3&!<*+.3*<=*(01&1)3/!'0+/''! +/';1&'/!<1);*+/.!01!0+*.3031&*9!/:/+<3'/?!@2/!;+3)*+,!;(+;1'/!15!023'!'0(.,!3'!01! ./0/+)3&/!02/!3&./;/&./&0!+/';1&'/'!15!ABC!*&.!1:3.*03>/!'0+/'!5191D3&4!*&! *<(0/!81(0!15!6+1'730!<1);*+/.!01!*!2342=3&0/&'30,!3&0/&'30,!EFGH!15!AB!)*:I!81(0! 15!0+/*.)39!/:/+<3'/?!!@2/!0+/*.)39!;+101<19!D39!8/!3&<9(./.!3&!02/!'0(.,!51+! <1);*+*03>/!;(+;1'/'?!! SIGNIFICANCE OF STUDY In terms of widespread popularity, CrossFit is a relatively new style of training. CrossFit is perhaps best described as high-intensity power training (HIPT) due to the physicaly demanding and time component-based workouts. Very litle research is currently available on CrossFit despite its popularity and unsubstantiated claims. The occurrence of physiological alterations following an acute bout of high- intensity exercise is wel established (2, 15, 29, 45, 55). Studies show that post exercise alterations result in increased susceptibility to untoward cardiac events as wel as damage to celular components (1, 4, 20, 31, 57). Heart rate variability (HRV) and oxidative stres are two independent physiological markers commonly used to evaluate alterations of cardiovascular activity and celular stres following acute bouts of exercise. Because of the lack of knowledge surrounding the high-intensity programing of CrossFit, further research and investigation of the physiological responses that occur following an acute bout is warranted. Therefore, it is the primary purpose of this study to investigate the ! J! alterations of HRV and oxidative stres following an acute bout of CrossFit and compare the alterations to that of a traditional high-intensity treadmil bout. ASSUMPTIONS The following asumptions were made: that al subjects are truthful in regards to inclusion criteria; that training state wil alow subjects to perform the bouts of exercise at the necesary intensity to elicit a physiological response; that HRV and oxidative stres markers are sensitive to this type of physiological stres. Furthermore, the use of VO 2max wil be used as a tool to predict CrossFit performance and therefore asist in the selection of subjects to beter homogenize the population. The asumption being that VO 2max wil acurately predict performance, such that lower VO 2max would predict lower number of rounds completed in CF and higher VO 2max would predict a greater number of rounds completed in CF. LIMITATIONS There are several limitations with the following studies. The first limitation is the training status is not controlled in this study. Increased tolerance to exercise stimulus has been wel established in participants who engage in regular physical activity (55). Secondly, outside cafeine and dietary supplementation (i.e., vitamins) restriction prior to the exercise bouts, diet is not controlled. Thirdly, this study wil only involve male subjects and wil not alow a determination of gender specific alterations or diferences in physiological responses in regards to a CrossFit workout. Lastly, the bout of CrossFit selected wil not fully represent CrossFit training as a whole due to the continuously varied nature of the programing. Therefore, the findings of this study wil be limited and only relate to CrossFit style workouts that are similar in duration and intensity. ! K! STATEMENT OF RESEARCH OBJECTIVES The overal objective of this research wil be to provide a beter understanding of HRV and oxidative stres following an acute bout of CrossFit. Furthermore these responses wil be compared to a traditional modality of exercise (treadmil running). This bout wil be a traditional high-intensity bout of treadmil exercise used as a comparative control. The specific objective of the first study is to determine the efect of a single bout of CrossFit and the high-intensity treadmil bout on the following markers of cardiac- autonomic modulations: Cardiac-Autonomic Modulations (HRV) a. The time domain measure of root mean square of the standard deviation of N-N intervals (RMSD) b. The frequency measure High Frequency measures within the recorded power spectral density (HF) The specific objective of the second study is to determine the efect of a single bout of CrossFit and high-intensity treadmil bout on the following markers of oxidative- stres responses within blood serum samples obtained: Oxidative-stres: c. Oxidation of Protein: Protein Carbonyls (PC) d. Oxidation of Lipids: Lipid Hydroperoxide (LOH) e. Antioxidant Capacity: Trolox Equivalent Antioxidant Capacity (TEAC) f. Antioxidant Potential: Ferric-reducing Antioxidant Potential (FRAP) ! L! ! CHAPTER II LITERATURE REVIEW OVERVIEW CrossFit is a relatively new form of ?high intensity power training? (HIPT) that has been growing in popularity within the mainstream. However, very few published scientific studies have examined this form of exercise empiricaly. As such, the current study wil measure blood markers of oxidative stres and cardiac-autonomic modulation before and after an acute bout of CrossFit. The remaining sections contained within this chapter wil provide a detailed description of CrossFit, cardiac-autonomic control, and oxidative stres. CROSFIT High-intensity interval training or ?HIIT? has become a popular form of exercise within mainstream. HIIT exercise combines multiple short bouts of high intensity exertion (i.e. above anaerobic threshold) with brief periods of complete rest or active recovery repeatedly for a prescribed period of time (e.g., 2-minute work intervals with 1- minute recovery intervals for 30 minutes) (39). Numerous studies have examined the efectivenes of HIIT and have demonstrated significant gains in aerobic capacity (25, 32, 39, 68). ! M! Recently, a form of HIIT training has emerged that removes scheduled rest periods and incorporates ?power? training within its format and has been termed ?high- intensity power training? (HIPT) (63). The goal of HIPT is to complete a prescribed amount of work as fast as possible. In other words, participants perform high-intensity, multiple joint resistance-based movements while maintaining a high power output (63). The lack of prescribed rest periods, the maintenance of a high power output, and the time-oriented goals (i.e., completing the overal workout as quickly as possible) are the major components that distinguish HIPT from HIIT (63). CrossFit is a form of HIPT that atempts to train a wide spectrum of physical fitnes components within one exercise program. Even though CrossFit is a newer approach to exercise, it is growing rapidly in popularity around the world (Smith et al), but has yet to gain footing within the scientific literature. For example, a recent (1/29/2014) Google search of ?Crossfit exercise? yielded over 14.2 milion results. However, a PubMed search of the same phrase yielded only 5 scientific studies. Therefore, it is obvious that much of the growth of CrossFit is based on unsubstantiated claims. The following five paragraphs provide an overview of the CrossFit standards and methods of programing. Please note that the information is referenced directly from CrossFit material (i.e., references 25 and 26), which is not validated by empirical evidence. Therefore, it is within this chapter only to discuss the principles behind their methodology. Discussion from CrossFit material: ! N! Theoreticaly, the foundation of CrossFit training began with the objective to best prepare an athlete for any physical contingency and to prepare them for the ?unknown and unknowable? (71). The programing is based on enhancing an individual?s competency across 10 general physical skils (70) that CrossFit state as the following: Cardio Respiratory Endurance, Stamina, Strength, Flexibility, Power, Speed, Coordination, Agility, Balance, and Acuracy. CrossFit training purportedly achieves fitnes acquisition across these skils by utilizing continuously varied, high-intensity, functional movements during each workout (71). The program combines multi-joint resistance exercises (e.g., deadlift, squat, power clean, and shoulder pres), gymnastic-based exercises (e.g., Olympic ring work, hand stand, and paralel bars), and traditional endurance-based modalities (e.g., running, rowing and biking). There are several variations and diferences betwen CrossFit workouts. The only consistent variable is the goal of moving weight for the heaviest load or as fast as possible in the given period of time (i.e., HIPT training). The daily CrossFit workout, as presented by CrossFit Head Quarters, is commonly referred to as the, ?workout of the day?, or ?WOD? (63). The WOD varies from day to day and typicaly includes a combination of the exercise listed above. However, the primary focus of a particular CrossFit workout may be categorized as mainly gymnastics based, Olympic lifting based, or ?metabolic conditioning? based (e.g., endurance). Each workout requires diferent physical demands and therefore varying the workout intensities. Some WODs are used as ! F! performance benchmarks and are typicaly named after a girl: e.g. ?Fran? and ?Cindy?. Two popular styles of CrossFit WODs are the ?clasic triplet? and as many rounds as possible (or AMRAP). The clasic triplet is a three round workout with the repetition scheme of 21-15-9. An example would be ?Fran?, which consists of a ?thruster? (i.e., an exercise that involves the front squat and pres) followed by pull-ups. Al repetitions of the first exercise must be completed before beginning the next exercise. In other words, 21 thrusters and pull-ups must be completed before moving on to the round of 15, and so on. The overal goal of this type of WOD is to complete the prescribed exercises and reps as fast as possible; no rests are scheduled or required. The goal of an AMRAP is exactly as the name implies, to complete as many rounds of a given exercise(s) as possible within a given time period. An example of an AMRAP would be ?CINDY?, which involves completing as many rounds of 5 pull-ups, 10 push-ups, and 15 air squats in 20 minutes. The goal is to perform as much work as possible in a giving time period. Most CrossFit facilities create and post their own WODs. However, CrossFit headquarters posts a new WOD everyday on the Internet (ww.crossfit.com) for anyone to atempt. Further examples of WODs and their potential variations can be examined in Table 1. Proponents of CrossFit claim that the rationale of program design is based off empirical data and is ?science-driven? (70), which is partialy true, in that CF paralels ! "G! wel quantified forms of exercise such as HIIT and Circuit training (5, 6, 30, 37?40, 44, 68). However, as previously stated only one published study has been performed on CrossFit. Smith et al. determined the efects of a CrossFit-based 10-wek program on aerobic fitnes and body composition in apparently healthy adult subjects. The results of the study revealed a 12% and 10% improvement in VO 2max and a 4.2% and 3.4% reduction in body fat % in male and female participants, respectively. Obviously, the study demonstrated a positive adaptation in aerobic fitnes and body composition after 10 weks of CrossFit training. Smith et al. (63) provides a glimpse into the possible benefits of chronic exposure to CrossFit. However, for being the first of its kind, this study did not demonstrate the physiological changes of an acute bout. Furthermore, there is no comparison to a traditional bout of exercise, e.g. treadmil running. Even though Smith et al. provides a novel first step, the overal lack of scientific data along with the drastic increases in the global popularity of this publicly acesible program warrants further investigation. CARDIAC ACTIVATION The heart is a complex organ that requires the synchronization of several tisues and physiological proceses to properly function. The cardiac muscle fibers are responsible for the contraction of the heart. These fibers share many similar properties as skeletal muscle fibers in that they are striated, contain contractile proteins, require calcium for muscular contraction, and can control the amount of force production (29, 55). Despite these similarities, cardiac muscle fibers have several important and distinctive diferences. In many respects cardiac tisue is expresed as a biochemicaly and physiologicaly unique fiber type as compared to skeletal muscle fibers. ! ""! Furthermore, unlike skeletal muscle fibers, cardiac fibers are more homogeneous in composition. Cardiac fibers contain a large number of mitochondria and are more oxidative than the highly endured type I skeletal muscle fiber. Another physical distinction is the short length and tight series formation of the fibers, connecting each other through intercalated discs (55). The intercalated discs alow the transmision of an electrical impulse from fiber to fiber, which is important when considering the depolarization of the cel. The intercalated discs create a leaky membrane, alowing ions to cross from one another. The importance of this characteristic is when one cel contracts, al interconnected cels contract. The unique functional arrangement of the discs can be referred to as syncytium, the firing of al cels as a single unit (29). For proper contraction of the heart it is vital that syncytium be under some form of control or cadence. It is the function of the sinoatrial node (SA node) to set the cadence for myocardial depolarization. The SA node is comprised of highly excitable tisue that posses a lower resting membrane potential then the other myocardium tisue, which alows it to depolarize first. Heart rate is controlled by both parasympathetic and sympathetic stimulation. The rate of depolarization of the SA node of the heart (pace maker) is determined by the fluctuations and dominance of parasympathetic/sympathetic activity (21, 29). The dynamics of this interplay becomes a pivotal role on resting heart rate and are sensitive to changes in the internal environment. Parasympathetic fibers (vagus nerve) are supplied by the cardiovascular control center in the medulla oblongata. The vagus nerve innervates the SA and atrioventricular (AV) nodes of the right atrium. When activated the nerves release acetylcholine on to ! "#! muscarinic-cholinergic receptors of the SA and AV nodes, reducing their depolarization and decreasing heart rate (55). Vagal activity is the typical stimulation at rest and is referred to as parasympathetic tone (29). Regulation of HR during resting periods is acomplished through increased or decreased parasympathetic activity alowing HR to increase or decrease respectively. Sympathetic fibers (cardiac acelerator nerves, T-1 through T-4) are also supplied by the cardiovascular control center (29). The cardiac acelerator nerve innervates the beta-receptors of the SA and AV nodes as wel as the beta-receptors on the ventricular myocardium. When the sympathetic nerves are stimulated they release epinephrine (E) or norepinephrine (NE) on to the receptors of the SA and AV nodes causing them to depolarize more rapidly, resulting in a more rapid HR. Furthermore, stimulated beta- receptors on the ventricular myocardium cause an increase in contractility (or strength of contraction), which increases the stroke volume. The combination of beta-receptor activation results in an increased cardiac output. Any increase in HR below 100bpm is primarily responsible to a withdrawal of parasympathetic activity, while anything above 100bpm is typicaly due to increase in sympathetic activity (55). The continuous changing of the parasympathetic/sympathetic ratio in response to physiological stimuli is known as sympathovagal balance and can be examined through the measure of heart rate variability. Heart rate variability is a commonly used measure of the autonomic nervous systems control of the heart. HEART RATE VARIABILITY The autonomic nervous system (ANS) plays a key role in regulating the cardiovascular system (21, 29, 55). The sympathetic and parasympathetic branches of ! "$! the ANS harmoniously increase and decrease, respectively, the heart?s rate and activity. The degree of influence of these systems is continuously changing and is often described as sympathetic-to-parasympathetic ratio or sympathovagal balance, which leads to oscilations in heart rate. Therefore, heart rate variability (HRV) is commonly used in research to noninvasively ases ANS modulation of the cardiovascular system (12, 21, 22, 27, 69). Measurement of HRV is acomplished through the quantification of the variations that occur betwen succesive R-R intervals on an electrocardiogram (ECG). The ?R? of the R-R interval is the peak of the QRS complex during ventricular depolarization. The R-R interval itself is the distance or time betwen QRS complexes (21). In other words, HRV measures the variation of time in betwen consecutive heartbeats. MEASUREMENT TECHNIQUES Time domain analysis of HRV involves transforming the ECG segment in which HRV is analyzed to a graph caled a tachogram (21). The time domain is a statistical calculation of adjacent R-R intervals. The y-axis of the tachogram represents the time (usualy in miliseconds) betwen each consecutive N-N interval, while the number of total beats is represented on the x-axis (21). There are a large number of time domain measures used in the literature (21). However, the root mean square of the succesive diferences (RMSD) is one of the more common time domain parameters, as it appears to be an acepted marker for parasympathetic modulation (21, 51?53, 65). Traditionaly, time domain measures were reported to require long-term recordings, such as a 24-hour period, and relatively stationary conditions (21). However, Tulppo (69) demonstrated that time domain measures in short recording periods (e.g., around 5-minutes) ! "J! demonstrate a higher sensitivity to cardiac-autonomic modulations. Therefore, this disertation wil use RMSD as the time domain measure of HRV for various 5-minute segments recorded during the pre- and post-exercise conditions. The frequency domain is another method to analyze HRV that is often coupled with time domain measures in research (15, 21, 54). However, this method of HRV is more complex, yet has been suggested appropriate to use in short-term (i.e., ~5-minute) recordings (21). The frequency domain utilized complex algorithms such as the fast Fourier transformation to create a power spectral density. From the power spectrum, the power of the entire waveform (0.00 to 0.40 Hz) is evaluated as Total Power (TP). The power spectrum may also be characterized at diferent frequencies, such as very low frequency (VLF, <0.04Hz), low frequency (LF, 0.04-0.15Hz), and high frequency (HF, 0.15-0.40Hz) power. What the power spectrums represent are often debated (7). There appears to be litle evidence to explain what VLF represents (21). Though LF is often reported in research, its meaning is stil unknown as it has been shown to be influenced by parasympathetic and sympathetic modulation, as wel as baroreflex sensitivity (7). However, HF is widely acepted as the representative marker of parasympathetic activity and controlled almost solely by the vagus nerve (31), as atropine and vagal nerve removal abolishes this marker (7, 27). Furthermore, the ratio betwen LF and HF (LF:HF) is commonly used to represent sympathovagal balance (21, 31). However, Bilman discusses several factors that demonstrate the unreliability of the LF:HF ratio as an accurate marker of symapthovagal-balance (7). In order for the LF:HF ratio to be considered an acurate measure of the balance of activity betwen sympathetic and parasympathetic nervous systems the following three ! "K! asumptions need to be made. First, SNS and PNS must be the major/exclusive factors involved in LF and HF frequency, respectively. Second, changes in one branch of the ANS must be met with corresponding changes in the other. Third, a linear relationship must exist betwen cardiac and ANS activity. Acording to Bilman, the SNS in not the major contributor to LF frequency (7). Furthermore an alteration in one branch does not directly correspond to changes in the other as the relationship betwen ANS and cardiac activity is complex and non-linear (7). Therefore, the asumptions necesary for LF:HF ratio to be considered acurate do not remain consistent, decreasing the reliability of that marker (7). The measurement of LF and HF components are often made in absolute values of power spectral density (i.e., in miliseconds squared), which represents the total amount of time the heart spent in each power domain. Each frequency domain is also normalized to acount for the total power and VLF weight by dividing either LF (LFnu) or HF (HFnu) by (total power-VLF) and multiplying by 100. Furthermore, HRV data within research studies often violate the asumption of normality and is naturaly logged transformed (ln) (65). PHYSIOLOGIC MECHANISMS OF HRV The direct relationship betwen HRV and parasympathetic and sympathetic activity is known. Furthermore, it is known that exercise increases the sympathetic response in the body (29, 31, 55, 69). Exercise is related to a decrease in RMSD and HF, a phenomena typicaly observed during exercise (11, 27, 31, 49). Increasing sympathetic activity and parasympathetic withdrawal results in a maximum loss of variance and no further alteration can be examined (e.g., total abolishment of HF) (13, ! "L! 14). However, there are reports where HF levels increased during exercise despite sympathetic activity, which is believed to be due to ventilation frequency and excitation of the vagal nerve (50, 66). The rate of which HRV returns to normal is also an indication of parasympathetic control of the cardiovascular system (11). There are several physiological reasons why exercise alters HRV and its recovery such as: parasympathetic withdrawal, circulating sympathetic nervous activity increases, hormones increase, breathing rate, and exercise induced increases in blood presure (29, 55). Of the aforementioned, the most obvious components that are responsible for alterations of HRV post exercise are changes in parasympathetic and sympathetic activity (21, 66). Previous sections detail how parasympathetic and sympathetic control work to increase or decrease heart rate. Parasympathetic-mediated changes in heart rate occur more rapidly than sympathetic-mediated efects (29, 66). Meaning that when control is parasympathetic dominant heart rate wil fluctuate more readily due to its increased sensitivity, creating more variability betwen beats (21, 66). Sympathetic dominance results in a rapid, but more consistent/les varied heart rate, and subsequently a decreased HRV (21, 66). The diferences in sensitivity of parasympathetic vs. sympathetic reaction may be caused by several factors: kinetics of ! 1 -adrenergic vs. muscarinic receptors, diferent kinetics of adenylate cyclase vs. phosphodiesterase, or the fast opening parasympathetic K ach channels (66). As the duration of a submaximal exercise increases or the intensity of a maximal bout of exercise increase so does the level of circulating hormones of epinephrine (E) and norepinephrine (NE), aldosterone, and angiotensin II (55). Increased adrenal hormones ! "M! such as E and NE are wel known to increase heart rate and contractility and subsequently decrease HRV. Decreases in blood presure and glucose may also contribute to increased levels during exercise, through the increased activation of the sympathetic nervous system (55). Circulating levels of E and NE remain elevated following removal of sympathetic activity following bouts of exercise. Aldosterone and angiotensin II increase linearly with exercise intensity and exert their efect on blood presure resulting in retaining water from the kidney and vasoconstriction respectively (66). Respiration plays a role in parasympathetic activity because the frequency is driven by the vagus nerve (50, 66). Respiration has been shown to cause fluctuations in the heart rate in a proces caled respiratory sinus arrhythmia (66). It is believed that exacerbation of respiratory sinus arrhythmia during exercise may be the cause increases in HF (50, 66). Further research is needed in regard to this relationship. HRV AND EXERCISE The measurement and interpretation of HRV originated clinicaly as a pragmatic instrument to predict untoward cardiac events (1, 21, 31, 69). Depresed HRV; that is, les variation of time betwen consecutive heartbeats, has been related to disease and increase risk for untoward cardiac events in clinical and non-clinical populations (21, 31, 69). However, recent applications involved the measure and analysis of HRV prior to or following bouts of exercise. The rebound of vagal activity, as measured through HRV, is believed to be an indirect measure of the bodies return to homeostasis (65). Furthermore, the recovery of HRV mirrors that of physical recovery and performance, becoming a viable tool in gauging physiological stres and the prescription of exercise (18, 36, 51?53, 65). ! "N! The parasympathetic nervous system plays a large role in recovery of HRV after physical activity. Goldberger et al. (27) demonstrated this relationship during exercise recovery through the use of the parasympathetic blocking drug atropine. Subjects participated in a maximal efort cycling bout and recovered in a seated position for 5min to record HRV. When the subjects were given the drug there was no recovery of time domain measures (SDN, Root Mean Square of the residual (RMS), RMSD). In contrast, there was a significant recovery of RMS and RMSD within the 5min recovery when the subjects were not provided with atropine. These findings demonstrate the role of the parasympathetic system in recovery. Furthermore, initial recordings of HRV post exercise were approximately the same as the parasympathetic blunted group, demonstrating the extent of parasympathetic depresion following exercise (27). Several other studies confirmed these findings of decreased HRV post acute exercise, revealing that the extent and duration of HRV depresion is related to the type, range, and duration of activity (15, 31, 69). Exercise intensity plays a role in alterations of cardiac autonomic modulations. In a study by Parekh and Le, subjects performed isocaloric treadmil bouts at 50% and 80% of VO 2reserve . The findings indicate a reduction of HRV occurs following both bouts of exercise. However, HRV was significantly lower following the bout of 80% VO 2reserve . SDN showed a similar trend with overal reduction post exercise, but the greater reduction was observed in the 80% intensity sesion. Frequency domain measures showed a reduction of TP, HF and LF in the 80% intensity sesion. HF measures were significantly reduced only during the first time point of the 50% intensity sesion. ! "F! Normalized LF (LF/[TP-VLF]x100) and LF/HF ratio increase was only observed in the 80% intensity sesion. The findings of a study performed by Buchheit et al. supports Parehk and Le in that they observed similar decreases in HF markers for high intensity exercise (~105% of final running speed at VO 2 max (V IFT )) and maintenance of HF markers following moderate intensity exercise (65% of V IFT ) (15). However, the time domain RMSD demonstrated no depresion in the moderate intensity trial, unlike what was observed in the Parekh and Le study. This may be in part due to diferences in protocol or diferences in time domain measurement techniques. Despite the findings of the aforementioned studies that have observed an overal reduction of HRV markers post acute exercise, Pober et al. demonstrated that markers of HRV might increase following a bout of submaximal exercise. Eleven healthy college- aged males participated in a 60min cycling bout at 65% VO 2peak . Measurements of HRV were obtained while subjects were in a supine position and breathing at a rate of 12bpm; time points taken were pre, 1, 3, 6, and 22hours post exercise. The time domain measures of SDN and RMSD yielded no significant changes post exercise. However, a positive numerical, but statisticaly insignificant trend was observed in pNN50 with increases at time points 1, 3, and 6. Frequency measure showed increases in HFNU at al time points following exercise. Similarly, there was a decrease in LFNU and LF:HF values. The review of the aforementioned studies suggests the overal mechanisms of control/recovery of cardiac autonomic modulation difer betwen maximal and submaximal exercise (15, 49, 54). ! #G! A large amount of the literature on exercise and HRV is focused on endurance exercise. However, resistance based exercise has been shown to afect cardiac autonomic modulation as wel (18, 31, 62). For instance, Hefernan et al. (31) examined HRV recovery following aerobic and resistance training modalities. Fourten moderately active males performed a resistance bout of exercise of 3x10 (10RM) and a 30 minute aerobic cycling bout at 65% VO 2peak , in random order. These findings suggest that HRV was depresed in both groups but the resistance bout had a more dramatic efect. Furthermore, Rezk et al. (62) where resistance exercise bouts were examined by intensity. Two exercise sesions (40%[E40%], and 80% [E80%]) performed a series of resistance exercises (E40%=3x20, E80%=3x10) and rested in the seated position to record ECG. The results of this study revealed a significant decrease in HF as wel as a significant increase in both LF and LF:HF ratio for both exercise intensity sesions. Interestingly, there was no significant diference betwen E40% and E80%. Though further research is needed, this demonstrates that within the mode of resistance exercise, intensity may not be the factor in alterations in HRV. As shown in the previously discussed studies, exercise intensity within aerobic/endurance exercises plays a major role in the magnitude of HRV depresion. Findings from several studies demonstrate the relationship betwen high intensity and a greater drop in cardiac-autonomic modulation (31, 35, 62). Modality plays a role in the degree of HRV depresion as wel. Resistance training depreses HRV to a greater extent than endurance training (31). However, when examining intensity betwen resistance bouts, the diferences appear to be negligible (62). ! #"! In conclusion, the application of HRV as a gauge of physiological stres and recovery has been demonstrated. Furthermore, the efects of exercise intensities and modalities on HRV difer and warrants consideration. The lack of empirical information concerning the exercise programing of CrossFit is evident. Therefore, it becomes imperative to examine the markers of HRV during recovery from a bout of CrossFit exercise. OXIDATIVE STRESS Oxidative stres results from free radical over production. Free radicals are atoms or molecules that contain one or more unpaired electrons, which makes them highly reactive (19, 56). Reactive Oxygen Species (ROS) is a term generaly used to describe a family of oxygen based radical species. Most pertinent to physiologic application are the ROS superoxide and hydrogen peroxide, respectively (56). ROS are a natural bi-product of numerous celular reactions and are routinely produced at diferent sites within exercised skeletal muscle tisue. Superoxide is the most common form of ROS and is produced at multiple enzymatic and non-enzymatic sub-celular sites both at rest and during exercise (56). Mitochondria: The mitochondria are the primary producer of aerobic generated ATP and thus a major source of muscular energy. The electronic transport chain within the mitochondria posseses protein channels that transport H + ions across the membrane in order to continue the electron further down the chain. As this occurs superoxide is produced in a stoichiometric, specificaly at complex I and III, relative to total metabolism (4, 56). ! ##! Sarcoplasmic Reticulum: Cardiac and skeletal muscle posses NAD(P)H oxidase enzymes within the sarcoplasmic reticulum. Using NADH as substrate, NAD(P)H oxidase wil produce superoxide as a bi-product (56). Transverse Tubules: Possesing a similar enzyme to NAD(P)H found in the sarcoplasmic reticulum, transverse tubules posseses NADP(H) oxidase. As depolarization occurs NADP(H) oxidase activity is increased and release superoxide into the cytosol as a bi-product (56). Other Sites: Quantified to a leser degree, ROS production also occurs from the plasma membrane, phospholipase A2-dependant proceses, and xanthine oxidase (56). The growing influence of these ROS in exercise scenarios is only now being revealed (60). There are several sites within the skeletal muscle capable of producing ROS. At rest production is minimal, while periods of exercise wil result in significant increases in production (43, 57). Major alterations of the internal environment due extreme exercise or physiological stres can result in severe levels of intracelular ROS and cel tisue damage or death (4, 43). Increased levels of ROS are responsible for the disruption of homeostasis by means of protein damage (contractile, transport, channel, structural, enzyme), lipid damage (damage to membrane structure and integrity), and damage to DNA (4, 43). The duration and magnitude of the increased levels of ROS are proportional to the oxidative damage incurred. In extreme cases this stres may lead to celular apoptosis/necrosis, while other cases may increase the likelihood of chronic disease. However, within the context of healthful exercise, ROS and the consequent oxidative stres are not considered ! #$! negative. The idea that exercise and disease are both asociated with oxidative stres is paradoxical at first glance. Resolving the mater, the oxidative stres asociated with exercise is transient, while disease causing oxidative stres is chronic (60). Recent evidence indicates that not only are ROS produced during exercise not detrimental to celular health, they?re esential to exercise induce adaptations (57). Moderate levels of exercise induced ROS production provide regulatory roles in the cels such as control of gene expresion, regulation of cel signaling and modulation of skeletal muscle contraction force (56, 57). In regards to gene expresion and cel signaling, lower levels of oxidative induced stres results in imediate activation of endogenous antioxidants coupled with a delayed expresion and up regulation of antioxidant enzymes (58). These adaptations result in physiological protection against oxidative stres. Exercise induced oxidative stres is directly related to the duration and intensity of the exercise that is taking place (43). As previously mentioned low to moderate levels of exercise induced oxidative stres provides positive adaptations, but extreme exercise may result in excesive oxidative stres levels are sometimes interpreted as acutely negative in terms of contractile performance (43, 56). OXIDATIVE STRESS AND EXERCISE Exercise elicits a transient oxidative stres response after an acute bout (10, 26, 33, 34, 41, 42, 46, 59, 61). Acordingly, both aerobic (8, 43) and strength (28, 34) exercise stimuli produce increases in identifiable oxidative stres biomarkers. The post exercise increases are often quantified in blood plasma/serum due to the availability of sample and the modern ability to quantify oxidized end products and antioxidant potential (17, 47). Bloomer et al. examined the change in protein carbonyl concentration in trained ! #J! men and women after three separate time trials on a cycle ergometer. Subjects performed cycling bouts at 70% of their VO 2peak for 30, 60, or 120 minutes, each trial occurring on a separate day. In order to ases plasma protein carbonyl levels 4 separate blood draws were taken; pre, 0post, 30min post, and 60min post. For both the 30 and 60min sesions protein carbonyl levels significantly increased at 0post and 30post with a peak change of 167% and 197%, respectively. Both returned to non-significant levels at 60post. In the 120min sesion al post exercise time points were significant with a peak change of 245% (8). Alesio et al. examined acute treadmil exercise at 80% of VO 2max and the efect of Vitamin C on oxidative stres. Oxidative stres was asesed by measuring the level of lipid peroxidation through TBARS. Subjects performed three bouts of exercise, the first a placebo trial, the second a one day vitamin C supplementation, and the third was a two wek vitamin C supplementation. The only group to elicit an oxidative response was the placebo exercise group with a 46% increase in TBARS (33). As discussed above, exercise induced increases in oxidative stres may sem to be counter intuitive without recent revelations that a wide variety of exercise derived benefits are due to ROS production (55). The up regulation of endogenous antioxidants post exercise makes this paradoxical relationship possible; however, too high of oxidative stres levels may lead to serious tisue damage (4, 56, 57). When examining the literature on oxidative stres and exercise a relatively smal amount exists on its relationship with resistance training. The existing research on resistance training and oxidative stres is equivocal with some studies having shown no oxidative response (9, 41), while others show significant increases after the bout (28, 34, 42). ! #K! Bloomer et al. investigated the oxidative stres response following sprints and repeated squats in anaerobicaly trained men. Each subjected performed repetitions of the squat until fatigue at 70% of their 1RM with 3min of rest in betwen sets. The number of sets was determined by the total amount of worked performed on a cycle ergometer 2 weks earlier, the average number of sets were 6.4? 0.28 (mean ? SED) while the number of repetitions averaged 40 ? 2. Bloomer measured oxidative stres through protein carbonyl and took blood measures at 5 diferent times points: Pre, Post, 30min, 24 hr, and 48 hr. Post-exercise measures of protein carbonyl did not significantly difer from pre measures (p=0.447), demonstrating that resistance training did not elicit an oxidative stres response (9). Similarly, McAnulty et al. showed no change in oxidative stres biomeasures in their study of resistance training and carbohydrate ingestion and its relation to oxidative stres. Oxidative stres was quantified through a gas chromatography mas spectrometry approach to measureusing F 2 -isoprstanes as the marker of stres. Subjects performed 10 diferent resistance training exercises consisting of 4 sets of 10 with the first set at 40% of 1RM and the following at 60% with 2-3minutes of rest betwen sets and exercises. Mean F 2 -isoprstane values exhibited no change after exercise compared to pre exercise measures, (p=0.193). In contrast to the findings of Bloomer and McAnulty, several studies have demonstrate a significant rise in oxidative stres after an acute bout of resistance exercise (10, 28, 34, 42). Hudson et al. compared the oxidative responses of acute strength based resistance exercise and acute hypertrophic based resistance exercise. In this later study, subjects performed two diferent back squat protocols, a hypertrophy and strength, separated by 1 wek. The hypertrophy protocol consisted of 4 sets of 10 at 75% of 1 RM ! #L! with 90sec of rest betwen sets. The strength program consisted of 11 sets of 3 at 90% of 1RM with 5min of rest betwen sets. Blood draws were collected pre, imediately post and 60min post for the measure of plasma oxidative stres markers protein carbonyl and lipid hydroperoxides. The hypertrophic and strength measures of lipid peroxidation showed no significant changes despite the upward drift in the 60min post hypertrophy measure, (p=0.156). Hypertrophic and strength measure of protein carbonyls showed significant increases only in the imediately post strength measure and in both 60min post measures (34). Goldfarb et al. examined resistance exercise and blood flow occlusion and its efects on glutathione and protein carbonyls. The non-occluded moderate exercise group performed bicep curls and calf extensions at 70% of their 1RM. Subjects completed al repetitions smoothly and through full rang of motion at a cadence of 0.67hz. Al sets progresed to failure with 1min of rest betwen each set. Blood draws were taken pre, post and 15min post to determine oxidative stres through protein carbonyl. The non-occluded group showed significant increases of protein carbonyls post and 15min post exercise (28). %&!'())*+,-!*9021(42!'1)/!'0(.3/'!./)1&'0+*0/!)3:/.!+/'(90'!1&!02/!1:3.*03>/! +/';1&'/-!*&!1>/+*99!+/>3/D!15!02/!930/+*0(+/!+/>/*9'!02*0!3&0/&'30,!*&.!)1.*930,!15! /:/+<3'/!D39!./0/+)3&/!02/!)*4&30(./!15!1:3.*03>/!'0+/''!/:;/+3/&/! '0+/''!+/';1&'/? MECHANISMS OF OXIDATIVE STRESS Like most reactions in the cel, ROS act on molecules by virtue of availability, and because al cel membranes are composed of proteins and phospholipids, they are ! #M! readily available for interaction. Oxidative modifications to proteins and lipids disrupt membrane stability and celular function. ROS induced free radical damage to proteins occur primarily in amino acids with exposed thiol groups (4). The damage to these amino acids results in decrease protein function, which can result in a variety of problems in that proteins serve several celular functions such as transport, receptor, enzyme activity, structural integrity or contraction (4). Oxidative damage of lipids is commonly referred to as lipid peroxidation. ROS wil react with polyunsaturated faty acids in membranes, resulting in propagation reactions creating more ROS as wel as continued lipid peroxidation (2). Oxidative damage to the lipid membrane results in altered membrane fluidity, increased permeability, and altered membrane function (2). Because cel membranes are composed of both lipids and proteins several sites for oxidative stres exist and membrane integrity wil be at risk. As the celular membrane loses integrity ionic dyshomeostasis occurs as evidenced by a rise in the concentration of intracelular levels of Na + , Ca ++ , and H + rise (2, 57, 58). These constituents wil eventualy lead to a noxious environment in the cel causing the release of mitochondrial cytochrome C, activating apoptosis (4). Quantification of the proces of oxidative stres includes biomarkers for protein modifications. Specific quantification of protein oxidation is commonly achieved through the measure of protein carbonyls found in the plasma (17). Exposed thiol groups in the amino acid are sensitive to ROS damage and serve as the location of carbonyl formation (4). Protein carbonyls are formed by a variety of oxidative stres mechanisms such as fragmentation and amine oxidation due to metal catalysis or by hydropochlorous acid (17). As the level of oxidants or ROS increase so does the likelihood of protein ! #N! damage. The most common and acurate lab technique to measure protein carbonylation is through enzyme-linked imunosorbent asay (ELISA). The protein carbonyl (PC) ELISA is an involved asay that requires several binding steps, however les plasma is required. The first step of this asay uses dinitrophenylhydrazine (DNP) as a primary binding agent to carbonyl groups (17). DNP specificaly binds to proteins that contain carbonyls, which flags oxidative damage. DNP alone is dificult to read and measure so streptavidin-linked horseradish peroxidase (hrp) is used as a marker that can be read through absorbance; however, Anti-DNP-biotin-antibody is required to atach to both DNP and hrp (17). In 1997 Buss et al. measured the sensitivity of the PC measurement for oxidative stres and found the PC ELISA asay to be both acurate and reliable (17). The protein carbonyl asay has been used succesfully in my previous work and is an established measure of protein oxidative stres in exercise applications (43). @2/!)/*'(+/!15!93;3.;/+1:3.*031&!3'!;/+51+)/.!02+1(42!*!>*+3/0,!15!*'*,'! 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WOD Exercises and Repetitions Prescribed Weights male/female Type Angie: 100 Pull-ups Body Weight For Time 100 Push-ups 100 Sit-ups 100 Squats Annie: Double-unders and Sit-ups: Body Weight For Time 50/40/30/20/10 Cindy: 5 pull-ups Body Weight (AMRAP)20 min 10-push-ups 15 squats Diane: Deadlift and Handstand Men (25lbs) For Time Push-ups: 21-15-9 Female (15lbs) Elizabeth: Clean and Ring Dips: Men (135lbs) For Time 21-15-9 Female (85lbs) Eva: 800 meter run Male (70lbs) 5 rounds 30 Ketlebel Swings Female (53lbs) 30 Pull-ups Fran: Pul-up and Thruster: Male (95lbs) For Time 21-15-9 Female (65lbs) Grace: 30 Clean and Jerks Male (135lbs) For Time Female (85lbs) ________________________________________________________________________ TABLE 1. Example CrossFit Workouts of the Day (WODs). For Time= complete as fast as possible, AMRAP= As Many Rounds As Possible. Prescribed weights are only for movements that require weight. ! $N! CHAPTER 3 ACUTE EXERCISE AND HEAR RATE VARIABILITY: CROSFIT VS. CONTINUOUS RUNING ABSTRACT CrossFit (CF) has become a popular form of exercise training throughout the world. Despite this, basic empirical evidence to describe physiological responses from CF, such as autonomic modulation of the heart, is lacking. The purpose of this study was to compare post-exercise HRV betwen CF and treadmil (TM) exercise. Ten men (age = 26.4 yrs ?2.7yrs) with at least three months of CF experience participated in the study. Aerobic power was determined with a maximal graded exercise test on a treadmil (GXT). On two separate occasions, each participant completed either the CF workout ?Cindy? (20min of as many rounds of 5 pull-ups, 10 push-ups, and 15 squats as possible) or a 20 min bout of TM exercise at 90% of GXT determined maximal heart rate. The exercise bouts were performed in a randomized crossover fashion. HRV was quantified through changes in the log-transformed square root of the succesive R-R diferences (lnRMSD) and high frequency power (lnHF), which was analyzed 5-min segments at 5- 10 min pre-exercise period (PRE), and during the post-exercise period at 15-20min (POST1), 20-25min (POST2), 25-30min (POST3), and 1hour (POST4). Significant time dependent decreases occurred following both CF and TM in lnRMSD and lnHF (p= 0.003) (p= 0.001) respectively. Trial dependent diferences were also observed in post- ! $F! exercise lnRMSD and lnHF measures, CF being significantly lower than TM (p= 0.002)(p= 0.000). Though a significant decrease in lnRMSD occurred after both exercise trials, lnRMSD at 1-hour post was not significantly diferent compared to pre following the TM bout (p= 0.172). However, lnRMSD at 1-hour post-CF remained significantly lower compared to PRE (p < 0.05). lnHF returned to baseline in CF and TM (p= 0.081)(p= 0.065). The results of this investigation demonstrated a depresion of HRV following CF compared to TM exercise. KEY WORDS: Heart Rate Variability, RMSD, HF, CrossFit, High-intensity INTRODUCTION Heart rate variability (HRV) is a noninvasive measure of the autonomic nervous (ANS) control of the cardiovascular system (8, 10, 19) and is basicaly defined as the oscilations that occur betwen consecutive R-R intervals that are derived from an electrocardiogram (ECG) (7, 10, 19). HRV is believed to be an indirect measure of parasympathetic activity (vagal tone) when asesed as the square root of the succesive R-R diferences (RMSD) and the high frequency (HF) of a power spectrum (10, 31). HRV application originated clinicaly in order to indirectly predict untoward cardiac events in both clinical and nonclinical patients, via chronic depresion (1, 2, 9, 11, 15, 31). Importantly, HRV is also depresed during periods of physical or emotional stres outside the presence of disease, and therefore can broadly be described as an indirect marker of physiological stres (2, 9, 13). Homeostatic recovery following physiological stres has become a focus in the applied sciences in an atempt to improve performance ! JG! and safer prescription of exercise (8, 17, 23, 29). The recovery of HRV (i.e., vagal rebound) post-exercise is used as a crude indicator of homeostatic recovery (4, 8, 17, 21? 23, 29). Exercise provides acute physiological streses to the body. For example, within seconds of dynamic muscle contraction, a withdrawal of vagal tone occurs, resulting in an acute period of depresed HRV that may last up to 72 hours post-exercise (8, 14, 20, 25). The magnitude and duration of post-exercise parasympathetic depresion depends on the intensity of the exercise, in that higher intensity exercise results in greater HRV depresion when compared to lower exercise intensities (8, 15, 16, 19). The recovery of HRV following acute bouts of exercise becomes important when considering future performance and prescription. Chen et al., found that recovery status mirrored vagal rebound following bouts of heavy resistance exercises (8). Furthermore, Kiviniemi et al., found that endurance programs guided by daily HRV measured upon waking used to determine the exercise intensity to be more succesful than standardized programing (17). A review by Stanely et al., examined the application of HRV as a tool to gauge the recovery status of an athlete for exercise prescription on basic cardiovascular exercise (29). Understanding the efect of exercise type and intensity on recovery is an important concept in HRV mediated programing (29). However, information on post-exercise HRV outside of endurance type exercise is lacking (29), and currently no empirical data is available on the high-intensity power training style known as CrossFit. CrossFit (CF) is a relatively new form of training that drasticaly warrants scientific study (27). The basic structure of a CrossFit workout is completing a combination of resistance and endurance exercise modalities as fast as possible, creating ! J"! a short duration and high-intensity bout. The popularity of CF has grown significantly over the past few years due to the short time commitment of each workout and frequent anecdotal reports of succes on the Internet. A Google search of ?CrossFit? and ?exercise? offers 14.2 milion results (1/29/14), while a scientific search on PubMed of the same key words offers only 5 results. This demonstrates the overal popularity of CF as wel as the lack of empirical evidence surrounding this form of training. The magnitudes of physiological stres caused by CF?s multi-modal and high-intensity exercise bouts are unknown. Due to the increasing popularity across the world, it becomes important to investigate this topic. It is the purpose of this study to evaluate exercise induced stres through the magnitude of parasympathetic depresion following a 20-minute bout of CF compared to a 20-minute bout of high-intensity treadmil (TM) running. A comparative analysis of post-exercise HRV wil provide a look at any existing diferences in modality. Modality has been shown to afect HRV diferently, most notability in Hefernan et al., where an acute bout of resistance exercise demonstrated a greater drop in HRV following resistance based exercise (15). This current study is novel in that it is the first to examine HRV following a bout of CF; furthermore, it is the first study to compare CF to a time and intensity matched bout of TM. It is hypothesized that the CF trial wil elicit a greater drop in parasympathetic activity as wel as a greater duration of parasympathetic depresion when compared to time-matched treadmil running. Methods Al data were collected in the Human Performance Laboratory (HPL), Auburn University at Montgomery (AUM). Each participant was required to arrive at the HPL ! J#! on three separate occasions; the first for familiarization of protocols and body composition measurements, followed by two separate occasions for data collection betwen 7am and 11am. PARTICIPANTS Auburn University and Auburn University at Montgomery institutional review boards approved this study. Participants were recruited through flyers and emails sent to local CrossFit establishments. Prior to participation, writen informed consent was obtained from each participant. Ten apparently healthy males participated in this study, descriptive data expresed as means ? SD can be sen in Table 1. Each participant followed CrossFit training for a minimum of three months. Al participants were of low risk for cardiovascular, metabolic, and/or pulmonary diseases as determined by PAR-Q and Health History Questionnaire. Participants reported not taking any prescribed medication during the time of the study. An inability to properly perform required movements, maximal oxygen consumption (VO 2Max ) under 40ml?kg -1 ?min -1 , or any symptom or contraindication of health resulted in exclusion from the study. Participants were informed that they could withdraw from the study at anytime. Participants were instructed to wear exercise clothing for testing, abstain from exercise 24 hours prior, and abstain from cafeine 12 hours prior to exercise testing sesions. MAXIMAL EXERCISE CAPACITY AND BODY COMPOSITION VARIABLES Maximal oxygen consumption (VO 2max ) and maximal heart rate (HR max ) was asesed during the first sesion through a graded exercise test (GXT) on a treadmil (Trackmaster, Newton, KS). Using Bruce Protocol, the workload during the GXT was increased incrementaly every 3 minutes until a maximal value was reached. Expired gas ! J$! (oxygen and carbon dioxide) fractions were continuously sampled at the mouth using a pneumotach, mixing chamber, and gas analyzers from a metabolic cart (ParvoMedics, Sandy, UT). During the test, heart rate was asesed continuously using a heart rate monitor (Polar Electro Oy, Oulu, Finland). Maximal heart rate obtained during this test was used to determine the target HR of the TM bout. Test termination required two of the following to occur: a plateau in VO 2 with increasing workload; respiratory exchange ratio (RER) of > 1.10; heart rate within 10 beats of age predicted maximum. Body fat percentage was asesed through Dual Energy X-Ray Absorptiometry (DXA) scan (GE Lunar Prodigy, Software version 10.50.086: GE Lunar, Corp., Madison, WI, USA). Prior to scanning, the DXA was calibrated using a standard calibration block. Participants were measured for height and weight then instructed to remove any metalic objects prior to scanning. Subjects were placed in a supine position and remained motionles during the scan. EXERCISE PROTOCOLS Exercise trial visits consisted of a 20-minute bout of either CF or TM, with pre and post period measurements of HRV through ECG. Exercise trials were randomized betwen CF and TM sesions. After resting ECG was acquired participants performed a standardized 5-minute warm up on the treadmil, rest for 1-minute then perform the exercise bout of CF or TM. Participants were not alowed to eat during the 1-hour recovery period. The study design can be sen in Figure 1. HR intensity (%HR max ) and rate of perceived exertion (RPE) scale numbered 1-10, with 1 being no exertion and 10 being maximal efort, were examined during this study to ensure similar efort betwen trials; results can be sen in Table 2. ! JJ! CROSFIT PROTOCOL The CrossFit named workout "Cindy? consists of as many rounds possible of 5 pull-ups, 10 push-ups, and 15 air squats in 20-minutes. The workout required that one complete al prescribed repetitions for the movement before moving on the next and to do so as fast as possible. Each movement was standardized for al participants. Pull-up form standards required that participant to start with arms locked out, pull their chin just above the bar, and return to the locked position. Push-up form standards required that participant to start with the arms locked out, lower the body to the ground until the chest touched, then return to the starting position. Air-squat form standards required that participant to start in the upright standing position, squat until hips past the knee, and return to starting position. Failure to achieve these standards resulted in the participant correctly repeating that movement. Upon completion of the workout participants were placed in a supine position for the designated periods of time, where post-exercise ECG was obtained to analyze HRV. HIGH-INTENSITY TREADMIL PROTOCOL To perform the TM trial, participants performed a minimum of 85% maximal HR obtained during the GXT. The target HR was determined via pilot study data not published. Participants ran for 20-minutes at a rate and incline that yielded a HR response within the target zone. Upon completion of the workout participants were placed in a supine position for the designated periods of time, where post-exercise ECG was obtained to analyze HRV. ANALYSIS OF HEART RATE VARIABILITY ! JK! For HRV asesment, participants were placed in a quiet, dimly lit room, and instructed to lie in a supine position on a comfortable examination table. ECG asesment was performed with a modified Lead II configuration using three Ag/AgCl electrodes and was interfaced with a Biopac MP100 data acquisition system (Goleta, GA). Three separate ECG recordings were obtained throughout each trial; Two, 10- minute segments with one at PRE and one at POST-60-minute, One 30-minute ECG segment imediately following the exercise bouts. The ECG recordings obtained in this study were divided into five 5-minute segments for analysis of HRV: the PRE time point measuring the last 5-minutes of the 10-minutes recording; the POST time points from the 30-minute post exercise recording, which was broken down into 3 POST 5-minute segments at 15-20, 20-25, and 25-30 minutes; and the 60-minute post-exercise ECG recordings examining the last 5-minutes of the 10-minute recording. Al ECG segments were visualy inspected for ectopic/non-sinus beats, which were replaced by the adjacent R-R interval when observed. Any segment containing three or more ectopic beats was excluded from analysis. Vagal activity was quantified through changes in the heart rate variability index of log-transformed square root of the succesive R-R diferences (lnRMSD) and log-transformed high frequency (lnHF) power, which was analyzed through 5-minute ECG segments. The markers chosen for HRV in this study were the time domain as the root mean square of the standard deviation of consecutive N-N intervals (RMSD), and frequency domain as High Frequency (HF) power (0.15-0.40 Hz). The transformation of ECG into time domain and Frequency domain components was done through specialized HRV software (Nevrokard version 11.0.2, Izola, Slovenia). In order to ases RMSD, the ! JL! ECG recordings were converted into a tachogram, which plots the succesive R-R intervals (y-axis) against the number of beats within the ECG (x-axis). From the tachogram the 5-minute segments were calculated for RMSD. Analysis of the frequency domain was performed through a power spectral analysis, which was completed by applying a fast Fourier transformation to the R-R intervals of the sampled ECG. RMSD and HF are sensitive markers of parasympathetic activity and have been used in several studies (3, 18, 21?23). RMSD is not significantly influenced by breathing frequency and is capable of measuring parasympathetic activity in a short period of time (23), making it a suitable marker for this study. The frequency domain has several components (i.e., VLF, LF, HF, LF:HF); however HF Is the only frequency marker that is widely acepted to acurately reflect vagal activity (2, 3, 6, 10, 12, 16). STATISTICAL ANALYSIS Subject HRV data was entered into SPS for statistical analysis. A Shapiro-Wilk test was used to determine normal distribution of HRV data. Because data were skewed, a natural logarithmic transformation was performed on the RMSD and HF data prior to further statistical analysis (raw data can be observed in appendix A). A 2 (Trial) x 5 (Time) repeated measures analysis of variance (ANOVA) was used to ases diferences from resting HRV to post exercise HRV in and betwen both CrossFit and Treadmil trials. A Paired Samples T-Test was used to further ases diferences betwen trial-to- trial time points, and pre to 1-hour post same trial time points. Statistical analysis was performed on SPS 19.0 (Chicago, IL). Statistical significance was set to " # 0.05. Data presented as means ? standard deviations. RESULTS ! JM! Al 10 participants of the study completed pre and post-exercise supine HRV analysis for the CrossFit and Treadmil trials. The markers of HRV are presented in lnRMSD (Figure 2a.) and lnHF (figure 2b.). The repeated measures ANOVA revealed that both CF and TM trails experienced a time dependant change in lnRMSD. For instance, lnRMSD dropped significantly in al time points when compared to PRE values following the bout of CF (p < 0.05). A Paired Samples T-test revealed POST 60- minute lnRMSD following CF remained significantly lower than PRE lnRMSD values (p < 0.05). Following the TM bout a significant, yet leser time dependant change in lnRMSD was observed (p < 0.05), which returned to near baseline values by POST 60- minute with no statistical diference from PRE (p = 0.17). A significant trial-to-trial diference was observed betwen CF and TM. Following a bout of CF a significantly greater depresion of lnRMSD occurred in each POST time point when compared to TM values (p < 0.05). A repeated measures ANOVA of the frequency domain revealed a significant time dependant decrease in lnHF following both CF and TM trials (p < 0.05). In both CF and TM POST 15-20, 20-25, and 25-30-minutes lnHF were significantly lower compared to PRE values (p < 0.05); however, both CF and TM returned to non-significant lnHF by POST 60-minute (p = 0.81, p = 0.65, respectively). A trial-to-trial comparison revealed a significant diference betwen the first three recovery time points with CF lnHF being significantly lower (p < 0.05). No significant diference was observed betwen CF or TM at POST 60-minute (p = 0.90). DISCUSSION ! JN! CrossFit is a relatively new style of training that has recently increased in popularity. To date, litle is known about the efects of a single bout of CF on HRV or how these results may compare to traditional exercise, such as continuous running. The magnitude and duration of HRV depresion may be related to cardiovascular strain following an acute bout of exercise (9, 10, 15, 29, 31). The magnitude of cardiovascular strain (i.e., dyshomeostasis) relates to the duration of recovery (29). Research in this area could have important implications related to the safe and efective prescription of exercise. Therefore, this investigation wil contribute to the current body of knowledge involving HRV following acute bouts of high-intensity exercise and short-term recovery. The purpose of this study to examine the afects of a CF bout versus a bout of time and intensity matched TM running on post-exercise HRV. We hypothesized that each trial would elicit an acute drop in both markers of HRV (lnRMSD and lnHF), but that CF would elicit a greater drop in the HRV parameters. The major findings of this study were that both CF and TM bouts resulted in a time and trial dependent depresion of HRV (lnRMSD and lnHF). Both trials resulted in post-exercise depresion of HRV markers were significantly diferent from resting measures. However, the CF trial demonstrated a greater depresion of HRV when compared to the bout of TM exercise. The POST 60-minute recovery time point for lnRMSD following CF remained significantly depresed compared to PRE lnRMSD parameters. In contrast, lnRMSD following TM and lnHF following CF and TM returned close to baseline levels by POST 60-minute. Control of cardiac function is acomplished though both branches of the autonomic nervous system: parasympathetic (vagus nerve) and sympathetic (cardiac acelerator ! JF! nerves) branches. The vagus nerve innervates the sinoatrial and atrioventricular nodes (14, 25). During the onset of exercise, vagal activity is removed resulting in the initial increase of HR up to 100bpm (14, 25). At the same time, working tisue activates muscle aferents through muscle metabolites and or mechano/baro/chemoreceptors of the heart and skeletal muscle (25). Muscle aferents signal the cardiac command center (CCC) to increase HR via vagal withdrawal and cardiac sympathetic stimulation (14, 25). At the completion of exercise, cardiac activity progresively returns to a resting state, which is majorly contributed by sympathetic withdrawal coupled with increased vagal outflow (7). Despite the return to resting HR, vagal activity remains blunted which can be observed through post exercise HRV (7, 12). The continued vagal depresion is a result of complex autonomic nervous system function and dynamics not yet fully understood (7, 30). Other researchers have found decreased lnRMSD and lnHF following acute bouts of exercise, which supports the findings of the current study (6, 13, 15, 19, 24, 26). The intensity of exercise influences the magnitude and duration of depresed HRV, and therefore becomes an important factor in sympathovagal balance (6, 19, 29). Parekh & Le examined diferences in intensity betwen 50% and 80% VO 2Reserve in treadmil running, finding that 80% produced a greater drop in lnHF and HFnu (19). Similarly, Buchhiet et al., observed a greater drop in RMSD and lnHF following high- intensity running versus sub-maximal running (6). Despite the observed influence of intensity within the current literature, this study found a greater depresion of lnHF and lnRMSD following CF compared to TM running even though HR intensities were similar. ! KG! The Trial dependant diferences observed in this study are in agreement with the hypothesis despite a similarity in intensity, which suggests a relationship betwen the exercise modality and the magnitude of depresion of HRV. Hefernan et al. (15) examined this relationship betwen a bout resistance exercises versus a bout of cycling. Interestingly, both bouts resulted in significant depresion of HF and HFnu. However, a greater depresion was observed following resistance exercise (15). Although there is some evidence to support diferences in modalities and their afects on HRV recovery (15), a lack of information regarding this relationship exists and further investigation is required. Though it is outside the scope of this study to find physiological reasons for the alterations of HRV, we can postulate on a few physiological responses related to the diferences betwen the trials. The CF workout, ?CINDY?, consisted of pull-ups, push- ups, and body weight squats, which by nature of the resistance-based exercise and the loads experienced by the muscle groups, would recruit more muscle mas than treadmil running (28). Increase muscle recruitment is asociated with raises in circulating epinephrine (28), which has been shown to decrease HRV (25, 30). Furthermore, the alterations in posture and redistribution of blood to active muscle groups from transition from one exercise to another throughout the trial likely presented a chalenge to hemodynamics and consequently increased the catecholamine response (5, 25). Although this may offer an explanation for the observed diferences found within this study, further investigation is necesary to provide a beter understanding. LIMITATIONS ! K"! Although this study was a novel step towards the beter understanding of HRV following a multimodal high-intensity exercise bout such as CF, it was not without limitations. The marker used to control for intensity (%HR max ) was similar betwen trials, but statisticaly diferent. The observed marker of intensity (RPE) also significantly difered betwen trials; however, psychological perception and influences may be responsible for these observed diferences. Future studies should examine diferences in muscle recruitment, blood lactate, and plasma catecholamine levels in conjunction to HRV in order to provide a comprehensive view of vagal activity and potential constituents of its alteration. Furthermore, the participants of this study were trained and experienced with high-intensity exercise, and may respond to this type of stimulus diferently than an untrained participant. Therefore, future studies should examine the afects of CF on HRV in untrained participants. CONCLUISON In summary, results show that the depresion and recovery of parasympathetic markers (e.g., lnRMSD and lnHF) were significantly greater and of longer duration (CF lnRMSD only) than treadmil running when compared to baseline values. Therefore, CF workouts such as the type examined in this study may provide a greater degree of physiological stres compared to high-intensity treadmil running at a continuous pace within trained participants. 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Cardiol. 43: 2278?2280, 2004. ! KK! TABLE 1. Participant Characteristics Characteristic Values ? SD Age (yr) 26.4 ? 2.7 Height (in) 69.8 ? 3.0 Weight (lb) 181.7 ? 8.2 Body Fat(%) 11.2 ? 7.6 VO 2max (ml?kg -1 ?min -1 ) 44.4 ? 16.2 Max HR (bpm) 183.6 ? 6.4 N=10 Means +/- SD ! KL! TABLE 2. Exercise Intensity (Perceived and %HR max ) 6minute 10minute 16minute 20minute p value RPE CF 5.4(?1.7) 6.4(?1.5) 7.6(?0.8) 9.0(?1.0) (p= 0.005) time TM 4.6(?1.4) 5.6(?1.2) 7.2(?1.2) 7.2(?1.5) (p= 0.007) trial %HR max CF 93.3(?3.9) 94.6(?2.9) 95.9(?3.0) 97.7(?5.9) (p= 0.005) time TM 89.3(?3.9) 92.9(?2.4) 94.2(?2.7) 93.6(?3.3) (p= 0.000) trial N=10 Means +/- SD. RPE scale 1-10 lowest to highest exertion. CF= CrossFit, TM= Treadmil. ! KM! Randomized, Crossover ! Figure 1. Study design. ECG samples were taken at designated time points, represented ECG icons. Samples taken before and after (randomized, crossover design) 20 minutes CF and TM bouts. ! Treadmil ! PRE 20 minutes of exercise IPE-30m 60-70min CF ?CINDY? ! ! KN! i!h! i!h! i!h! h! i! i! i! i! i! i! i!h! i!h! i!h! i! FIGURE 2. HRV markers before and after exercise, Means ? SD, n=10. A) LnRMSD CF and TM B) LnHF CF and TM. * significant from pre, # significant CF vs T. Dashed line = CF, Solid line = TM A B ! KF! Apendix A. Raw RMSD & HF PRE and POST Trials (ms 2 ) PRE 15-20 20-25 25-30 60-65 RMSSD CF 64.73 (?33.22) 7.21 (?2.60) 8.92 (?4.10) 10.26 (?4.5) 33.24 (?22.10) TM 65.24 (?27.40) 18.39 (?12.75) 22.83 (?16.14) 24.09 (?12.66) 55.72 (?37.90) HF CF 1670.35 (?1567.57) 25.67 (?44.23) 32.29 (?41.49) 36.12 (?37.38) 551.89 (?897.65) TM 1794.53 (?1674.76) 301.57 (?422.30) 264.19 (?292.98) 1532.38 (?2468.20) 36.16(?16.03) ! ! ! LG! CHAPTER 4 ACUTE EXERCISE AND OXIDATIVE STRES: CROSFIT VS. TREADMILL BOUT ABSTRACT Purpose: High intensity power training (HIPT), including CrossFit, is a global fitnes phenomenon. Despite increasing popularity, basic empirical evidence to describe physiologic responses including acute oxidative stres to CrossFit are lacking. Therefore, the purpose of this study was to examine the acute efects CrossFit on blood redox changes. Methods: Oxidative stres responses were compared to a traditionaly prescribed bout of high-intensity treadmil exercise in 10 male 26.4 yrs (+/- 2.7yrs) with at least three months of CrossFit experience. Participant VO 2max was determined prior to completion of the CrossFit workout ?Cindy? (20min of as many pull-ups, push-ups, and squats as possible) or a 20 min 90% HR max Treadmil bout as determined by graded exercise test. Treadmil and CrossFit works were performed in a randomized crossover fashion. Blood plasma was collected at 4 diferent time points for each trial: pre, post, 1hr post and 2hr post, to examine lipidperoxidation (LOH), Protein Carbonyl (PC) and antioxidant capacity. Results: CrossFit and Treadmill elicited a time dependent increase of LOOH in 1hr post (143% and 115%) and 2hr post (256% and 167%) respectively. PC demonstrated a slight increase IPI (5% and 0%) then a time dependent decrease 1hr post (-16% and -8%) and 2hr post (-16% and -1%) compared to IPE. Ferric Reducing ! L"! Antioxidant Power (FRAP) also demonstrated a time dependent increase within CrossFit and Treadmil bouts; post (25%, 17%), 1hr post (26%, 4.8%), 2hr (20%, 12%) respectively. Total Enzymatic Antioxidant Capacity (TEAC) showed a time dependent decrease in post (-10%, -12%), 1hr post (-12%, -6%), 2hr post (-7%, -11%) respectively. No trial dependant diferences were observed in any biomarker of oxidative-stres. Conclusion: CrossFit elicited an acute blood oxidative stres response that is comparable to a traditional bout of high intensity treadmil running; however, the blood plasma antioxidant response to Cindy exhibited diferences during recovery. INTRODUCTION Reactive oxygen species (ROS) are oxygen centered radical compounds that modify molecules within a cel, or tisue resulting in damage (3, 23, 27). ROS encompases free radicals, which are atoms or molecules that contain one or more unpaired electrons in their outer shel, which results in high reactivity. Oxidative stres is a physiological phenomenon that occurs when oxidative damage surpases antioxidant defense (8). The production of ROS occurs during numerous physiological proceses, including physical exercise, and thus is a naturaly occurring event in cels throughout the body (3, 10). A prolonged increase of ROS is central to dyshomeostasis through the modification of protein and lipids and is influenced by exercise (5, 7, 14, 23, 25). Moderate increases of ROS are beneficial if not esential for optimal physiological performance (27), while excesive amounts hinder performance, or in rare and extreme cases cause celular death (3, 27). As applied to exercise, oxidative stres is commonly quantified using blood biomarkers of oxidative damage and antioxidant defense capacity (18, 21, 23, 31). ! L#! The production of ROS is proportional to the intensity of the exercise or the fatigue inducing duration of the bout (1, 2, 5, 31). In this regard, the majority of prior exercise induced oxidative stres findings are derived from studies that utilize ?traditional? modalities of exercise such as running, cycling, and resistance training. However, the blood oxidative stres response to power-based exercise bouts that employ low duration and high intensity have yet to be fully examined. Among the novel low duration-high intensity exercise modalities that are yet to be understood relative to blood oxidative stres is high-intensity power training (HIPT). The increase in popularity of HIPT has skyrocketed over recent years due to the brief time requirement and purported succes in weight loss (33). The most popular form of HIPT is CrossFit, a program that trains a wide spectrum of physical fitnes components within one exercise program. CrossFit incorporates both resistance and endurance modalities within a single bout. In addition, participants complete a prescribed workout as fast as possible, creating a short duration and high-intensity bout. Despite the increasing popularity of CrossFit, basic empirical evidence is ultimately lacking in terms of various physiologic outcomes including oxidative stres responses. Therefore, it was the purpose of this study to examine the blood oxidative stres response following a single bout of CrossFit. To undertake this study, a comprehensive biomarker panel of blood oxidative damage and antioxidant defense measures was performed before and after an acute bout of CrossFit exercise. Furthermore, these findings were compared to more traditionaly prescribe high-intensity treadmil exercise matched for time in order to ases any diferences betwen modalities. METHODS ! L$! PARTICIPANTS Auburn University and Auburn University of Montgomery institutional review boards approved this study. Prior to participation, signed informed consent was obtained from each participant. Ten healthy males, age 26.4 yrs (? 2.7) weight 181.7lbs (? 8.2), height 69.8in (? 3.0), and body fat 11.2 % (? 7.6%) participated in this study. Each participant partook in CrossFit training for a minimum of three months. Al participants were of low risk for cardiovascular, metabolic, and/or pulmonary diseases as determined by PAR-Q and Health History Questionnaire. Participants reported not taking any prescribed or over the counter medication during the time of the study. Subjects were instructed to abstain from exercise 24 hours prior to each trial, as wel as cafeine, alcohol, and vitamin supplementation 12 hours prior. MAXIMAL EXERCISE CAPACITY AND ANTHROPOMORPHIC MEASUREMENTS Maximal exercise capacity (VO 2max ) and maximal heart rate (HR max ) were asesed during the first sesion through a graded exercise test (GXT) on a treadmil (Trackmaster, Newton, KS). Using Bruce Protocol, the workload during the GXT was increased incrementaly every 3 minutes until a maximal value was reached. Expired gas (oxygen and carbon dioxide) fractions were sampled continuously using a pneumotach, mixing chamber, and gas analyzers through a Parvo Medics cart (Sandy, UT). During the test, heart rate was asesed continuously using heart rate monitor (Polar Electro Oy, Oulu, Finland). Test termination required achievement of two of the following criteria: a plateau in VO 2 occurs with increasing workload; respiratory exchange ratio (RER) of > 1.10; heart beat within 10 beats of age predicted maximum. ! LJ! Body fat percentage was asesed through total body Dual Energy X-Ray Absorptiometry (DXA) scan (GE Lunar Prodigy, Software version 10.50.086: GE Lunar, Corp., Madison, WI, USA). Prior to scanning, the DXA was calibrated using a standard calibration block, and followed manufacturer?s procedures. Participants were measured for height and weight then instructed to remove any metalic objects prior to scanning. Subjects were placed in a supine position and remained motionles during the scan. Subject characteristics are presented in Table 1. OXIDATIVE STRESS EXPERIMENTAL DESIGN Al data were collected in the Human Performance Laboratory (HPL), Auburn University of Montgomery (AUM). Each participant arrived at the HPL on three separate occasions for data collection betwen the 7am and 11am. The study design is presented in Figure 1. Exercise trials were randomized betwen CrossFit and High-intensity treadmil sesions. Blood was sampled before and after each trial. After the pre blood draw participants performed a standardized 5-minute warm up on the treadmil, rest for 1- minute then perform the exercise bout of ?Cindy? or High-intensity Treadmil. Participants were not alowed to eat during the 2-hour recovery period. BLOOD SAMPLES AND STORAGE Blood samples were collected and asayed for biomarkers of oxidative damage and antioxidant potential at the following time points; before exercise (PRE), imediately post exercise (IPE), 1-hour post exercise (1-HP), and 2-hours post exercise (2-HP). Participants were in a seated position while the 10 mL blood samples were taken. Draws were taken via venipuncture through the antecubital vein and were collected in EDTA tubes (2 mL) and heparinized tubes (8mL). Heparinized tubes were ! LK! imediately centrifuged at 3000 rpm for 15 minutes, aliquotted, and stored in ultra-low freezer -80?C until subsequent asay. Hematocrit and hemoglobin were determined using EDTA tube whole-blood aliquots using a hematology analyzer (CelDyn 1800, Abbott Park, IL). Participants were instructed to abstain from food or beverage other than water during the post exercise period. CROSFIT PROTOCOL The CrossFit named workout "Cindy? consists of as many rounds possible of 5 pull-ups, 10 push-ups, and 15 air squats in 20-minutes. The workout required that one complete al prescribed repetitions for the movement before moving on the next and to do so as fast as possible. Each movement was standardized for al participants. Pull-up form standards require the participant to start with arms locked out, pull their chin just above the bar, and return to the locked position. Push-up form standards require the participant to start with the arms locked out, lower the body to the ground until the chest touched, then return to the starting position. Air-squat form standards require the participant to start in the upright standing position, squat until hips past the knee, and return to starting position. Failure to achieve these standards resulted in a repetition of that movement. Upon completion of the workout participants were placed in a seated position for subsequent blood draws. HIGH-INTENSITY TREADMIL PROTOCOL To perform the HITP trial, participants performed a minimum of 85% maximal HR obtained during the GXT. The target HR was determined via pilot study data not published. Participants ran for 20-minutes at a rate and incline that yielded a HR ! LL! response within the target zone. Upon completion of the trial participants were placed in a seated position for subsequent blood draws. OXIDATIVE STRESS BIOMARKERS Blood plasma samples were asayed for oxidative stres biomarkers, which are used previously (18, 23, 30). Two biomarkers for oxidative damage were chosen, Plasma lipid hydroperoxides (LOH) and protein carbonyls (PC). Two additional biomarkers were selected to examine antioxidant capacity, Ferric-reducing antioxidant potential (FRAP) and Trolox-equivalent antioxidant capacity (TEAC). Lipid peroxidation was determined through modified ferrous oxidation-xylenol orange asay (FOX) and reported as jmol/L (26). FOX measures the oxidation of ferrous ions to ferric ions by lipid hydroperoxide, which reacts with the ferrous-sensitive dye (xylenol orange). In the presence of lipid hydroperoxides this asay creates a complex that can be quantified spectrophotometricaly (26). Protein carbonyls were asayed using a commercialy available ELISA kit (Biocel, Papatoetoe New Zealand), protocols followed asay kit instructions and reported as jM/mg protein. Prior to analysis, individual plasma protein content was normalized using the methods of Bradford (1976). Antioxidant capacity was measured through the Trolox equivalent antioxidant capacity (TEAC) asay with modified methods form Re et al. (1999) and Vilano, Fernandez- Pachon, Troncosco, and Garcia-Parrila (2004) (32, 35). The principle of the TEAC asay is that oxidized 2,2?-azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid) is reduced via antioxidants in the blood plasma (9, 32, 35). Total antioxidant potential was measured using ferric-reducing antioxidant potential (FRAP) asay, which methods were adopted and modified from Benzie and Strain (1996)(4). TEAC and FRAP are both asesed ! LM! through colorimetric solutions spectrophotometricaly (4, 35). The biomarkers of oxidative stres selected for this study are sensitive to exercise application and have been used on several occasions by our lab and others (7, 18, 23?25, 30). The rationale for this comprehensive examination of the selected biomarkers of blood oxidative stres comes from the concept of the ?oxidative stres pecking order?. Acording the pecking order, oxidative stres related damage would not occur until after endogenous antioxidant defense has failed (i.e., water-soluble antioxidants and lipid-soluble antioxidants, respectively) (8). Due to this current understanding, the oxidative stres biomarker panel used in this study is sensitive to protein and non-protein water-soluble antioxidants as wel as lipid-phase antioxidants (9). Al biomarkers were normalized for plasma-volume changes that occurred following the exercise trials using the formulas based on the established protocols of Dil & Costil, normalizing plasma through hemoglobin and hematocrit levels compared to preexisting levels (11). STATISTICAL ANALYSIS Participant oxidative stres data was entered into SPS for statistical analysis. A 2 (Trial) x 4 (Time) repeated measures analysis of variance (ANOVA) was used to ases diferences from resting oxidative stres to post exercise values in and betwen both CrossFit and Treadmil trials. For the key dependent variables, a Mauchly?s Test was run to determine there was no violation of sphericity. Statistical analysis was performed on SPS 19.0 (Chicago, IL). A Tukey post hoc was used to determine significant mean diferences when indicated. Statistical significance was set to " # 0.05. Data presented as means ? standard deviations. RESULTS ! LN! Al participants completed CrossFit and Treadmil protocols. Mean anthropomorphic values can be sen in Table 1. In addition to monitoring HR for intensity, a rate of perceived exertion (RPE) scale numbered 1-10, with 1 being no efort and 10 being maximal efort, was used. Percent HR max and RPE were used to ensure a similar efort of intensity betwen trials; results are presented in Table 2. Oxidative damage biomarkers are presented for blood plasma lipid hydroperoxides (Figure 2a) and blood plasma Protein Carbonyl content (Figure 2b). Statistical analysis of lipid damage (LOH) demonstrated a significant time dependant increase in both CrossFit and Treadmil trials (p< 0.001), while no trial-dependent diference where observed (p= 0.623). Plasma LOH did not exhibited a significant increase after 1-HP for CrossFit and Treadmil, 143% and 115% respectively (p< 0.001). At 2-HP values increased further to 256% and 167% compared to PRE values (p< 0.001). A significant diference was also observed betwen 1-HP and 2-HP (p= 0.025). Analysis of mean plasma protein carbonyl, demonstrated a time dependent decrease (p= 0.001), but no trial dependant diferences (p= 0.245). A non-significant increase was observed IPE (p= 0.187) followed by significant decreases in both 1-HP (p= 0.001) and 2-HP (p< 0.001) in both trials, only when compared to IPE. Biomarkers for antioxidant capacity are presented as FRAP (Figure 3a) and TEAC (Figure 3b). Analysis of FRAP demonstrated a significant time dependant increase in both trials (p< 0.001), but not betwen trials (p= 0.080). FRAP concentration increased significantly in al post exercise time points IPE (p< 0.001), 1-HP (p=0.001), and 2-HP (p=0.004). TEAC unexpectedly showed a negative time dependant reaction in both trials (p= 0.00), while no trial dependant diferences were observed (p= 0.134). ! LF! TEAC significantly decreased in al post measurements IPE (p< 0.001), 1-HP (p= 0.001), 2-HP (p= 0.001). DISCUSSION The purpose of this investigation was to examine the blood oxidative stres response to an acute bout of CrossFit exercise and compare them to a more traditional Treadmil bout normalized for time and HR intensity. The marker used to control for intensity (%HR max ) was similar betwen trials, but statisticaly diferent when examining area under the curve; however, this diference is not believed to be of physiological significance. The observed marker of intensity (RPE) also significantly difered betwen trials despite the similarities; psychological perception and influences may be responsible for the observed diferences. The key finding of this study was that markers of oxidative stres did not significantly difer betwen trials; however, a time dependent increase in oxidative stres markers occurred in each. These findings suggest the oxidative stres response to CrossFit is proportional to the exercise intensity performed and as such the time-frame for the observed oxidative stres responses paralel prior observations using diferent exercise modalities (14, 17, 18, 21, 22, 30). Additional points of consideration are provided below. MARKERS OF OXIDATIVE STRESS While not measured directly in this investigation, the sources of oxidative stres in this study are multi-faceted. One of the most physiological abundant oxidants, superoxide, is widely produced in the body and is rapidly converted to other ROS (3). Based the on current literature, there are several known sites for superoxide production, which include mitochondrial respiration through complex I and III, catecholamine auto- ! MG! oxidation, and various oxidases (3, 10, 27). Exercise is a wel-documented facilitator of ROS production throughout the body (10, 27, 28). This study examined two types of high-intensity exercise bouts in the form of CrossFit and time-matched Treadmil running. These trials were matched by time and %HR max to produce a common level of exercise intensity. Despite diferences in modality and muscle recruitment, no diferences in redox reaction were observed betwen trials. These findings support recent works that suggests the oxidative stres response is more sensitive to exercise intensity than modality (31). The marker for lipid damage, lipid hydroperoxide (LOH), increased in a trial- independent fashion following each bout and confirmed that exercise induced oxidative- stres occurred. Previous works supports these findings in that increasing measures of LOH occur following several diferent exercise modalities such as hiking, cycling, and weight lifting (18, 23, 30). Quindry et. al., observed that rises in LOH occur independently of aerobic metabolism, and significantly rise following exercise that surpases lactate threshold (31). Therefore, it is more likely that the exercise intensity of the bouts, approximately 90% of HR max , was the driving force behind the lipid hydroperoxide production. Given that CF and TM were closely matched for intensity, the similar rise in plasma LOH is not surprising. The time dependent decrease in Protein Carbonyl (PC) was an unexpected result of the study. PC is a sensitive marker of ROS mediated protein damage and has been shown to increase in plasma after bouts of acute exercise of varying intensities (5, 14, 18, 24, 29, 30). However, the current observation is not the first of such instances where exercise does not result in an increase in PC (Quindry in review, (6). Bloomer et al. ! M"! observed no change in PC following an acute bout of squats or sprints, which interestingly trended towards a decrease (6). Similarly, Quindry et al. observed no change imediately post ecentric exercise; however, 24 hours following the sesion, a significant rise in PC was observed (29). The oxidative damage response of this study yielded mixed results, both expected and unexpected. LOH increased following the trials only after the first hour of recovery, while PC unexpectedly decreased following the trials. The lack of response in PC suggested that ROS mediated protein damage may not have occurred; however, the delayed PC response in the Quindry et al., study suggests that oxidative stres mediated protein damage may occur through a larger window of time, and therefore the magnitude of response in this study may not yet have occurred (29). These findings of discordant biomarker results also reinforce the need for a panel approach to quantify wide biological proceses such as oxidative stres. MARKERS OF ANTIOXIDANT DEFENSE In order to gain a comprehensive look at redox balance, both oxidative damage and antioxidant defense were examined. Findings from the current study included antioxidant defense through the antioxidant potential (FRAP), which is sensitive to aqueous- and lipid-phase anti-oxidants, and antioxidant capacity (TEAC), which is sensitive to protein and nonprotein sulfhydryl antioxidants (9). The importance of redox balance is ilustrated in a clasic concept referred to as the ?oxidative stres pecking order?. Acording the pecking order, oxidative stres related damage would not occur until after endogenous antioxidant defense has failed (8). Therefore, oxidative damage wil occur when redox balance is lost. ! M#! The antioxidant potential, ascorbate equivalent (FRAP), measures the antioxidant reducing ability in plasma and is commonly elevated in response to rising levels of ROS (4). The blood plasma biomarker FRAP increased significantly IPE and remained elevated throughout the time points following CrossFit and Treadmil protocols, which is an expected response due to the presence of oxidative-stres in both trials. A similar elevation of ascorbate equivalent was observed in a study performed by Quindry et al., where cyclists performed three separate climate conditioned (cold, warm, neutral) 60min protocols (30). Hudson et al. experienced mix results when examining FRAP levels following bouts of strength and hypertrophy exercises, only observing a significant rise 60min following hypertrophy (18). However, MaCnulty reported elevated levels of FRAP following ten separate resistance exercises in a interval scheme (21). The response to increased repetition scheme suggests that duration and intensity play a role in antioxidant potential. Uric acid, a by-product of Purine metabolism, has been shown to increase with elevated exercise intensity (34), which is important to note because the FRAP asay is primarily driven by uric acid (9), and therefore a potential explanation for the observed increase in post exercise FRAP. Antioxidant capacity measured through TEAC decreased significantly following both trials. A decrease in antioxidant capacity is an unusual occurrence in the presence of oxidative damage as wel as increased antioxidant potential. Several studies have demonstrated a concurrent increase of TEAC and oxidative stres (21, 23, 30), while only a few studies have observed a decrease in TEAC following exercise(12, 18). Hudson et al. observed a decline of TEAC 60 minute following strength-based exercise, while hypertrophy based resistance exercise resulted in an increase IPE (18). In untrained ! M$! participants Falone et al. observed a decrease in TEAC following a maximal treadmil test, in contrast they observed an increase in TEAC in trained participants (12). There was no physiological evidence or explanation for the decline in TEAC in these studies. The observed decrease of plasma TEAC and PC in this study may reflect a shift in plasma volume and density known as Haemoconcentration (16). In a postural study by Szalky et al., plasma density was measured through plasma albumin and globulin, which decreased during periods of haemoconcentration, resulting in a net loss of plasma protein (16). Haemoconcentration following bouts of high-intensity exercise results in no change in plasma protein concentration or in a few instances a decrease in plasma protein (13, 15, 19); however, shortly after haemoconcentration (times may vary up to 24-hours) plasma shifts rebound and result in a period of haemodilution, which increases plasma protein concentration (19). An additional phenomenon that may be responsible for decreased plasma protein concentration is post exercise proteinuria. During heavy exercise filtration fraction in the kidneys increase alowing proteins into the ultrafiltate or maximal tubular reabsorption resulting in consequential protein loss in urine (proteinuria) (20). The possibility of haemoconcentration and/or post exercise proteinuria are both potential factors for the observed decreases in PC levels following the trials and subsequently decreased TEAC. The TEAC asay is driven primarily through an albumin- based reaction, which contributes approximately 28% to the asay (9). Because both PC and TEAC are albumin-based asays, a decrease in plasma protein (i.e., albumin) may be related to the decreases observed both PC and TEAC asays. Further investigations of these relationships are needed. Summary and Conclusions ! MJ! CrossFit is a relatively new style of training that has become widely popular. To date, litle is known about the afects of a single bout of CrossFit on Redox reaction or how these results may compare to a time-matched bout of Treadmil running. High levels of free-radicals are known to cause celular damage, however moderate increases are responsible for several beneficial celular functions (27). The oxidative stres response to CrossFit was significant enough to ilicit lipid peroxidation and increase antioxidant potential, although this was not significantly diferent than a time and intensity matched bout of treadmil running. This indicates that CrossFit may be no more stresful than high-intensity treadmil running, within trained subjects. The amount of oxidative stres is directly related to the level of exercise intensity rather than the exercise modality, and therefore should be taken into consideration during the prescription of exercise. The lack of PC and TEAC in this study may be more of an indication of timing rather than a lack of oxidative stres and therefore, further research is necesary to determine the full extent of acute oxidative stres on CrossFit bouts. In summary, the findings of this study indicate that there were no statistical diferences in oxidative damage or redox balance betwen CrossFit and Treadmil running. Results also confirm that exercise intensity is a major contributing factor in the nature of the oxidative response as wel as the time course. These tests were performed on CrossFit trained individuals familiar to the stres involved with this type of exercise. Future research should examine untrained participants in order to gain a beter understanding of the physiological streses involved in CrossFit. Furthermore, because the redox time course was not evaluated beyond two hours into recovery, future studies should examine high-intensity bouts with a more extensive timeline. The implications of ! 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Participant Characteristics Characteristic Values ? SD Age (yr) 26.4 ? 0.9 Height (in) 69.8 ? 0.9 Weight (lb) 181.7 ? 2.5 Body Fat(%) 11.2 ? 2.4 VO 2max (ml?kg -1 ?min -1 ) 44.4 ? 5.1 Max HR (bpm) 183.6 ? 2.0 N=10 Means +/- SEM ! NG! TABLE 2. Exercise Intensity (Perceived and %HRmax) 6minute 10minute 16minute 20minute p value RPE CF 5.4(?0.5) 6.4(?0.5) 7.6(?0.2) 9.0(?0.3) (p= 0.005) time TM 4.6(?0.4) 5.6(?0.4) 7.2(?0.4) 7.2(?0.5) (p= 0.007) trial %HRmax CF 93.3(?1.2) 94.6(?0.9) 95.9(?1.0) 97.7(?1.9) (p= 0.005) time TM 89.3(?1.1) 92.9(?0.8) 94.2(?0.9) 93.6(?1.0) (p= 0.000) trial N=10 Means +/- SEM. RPE scale 1-10 lowest to highest exertion. ! N"! Randomized, Crossover ! Figure 1. Study design. Biomarkers of oxidative-stres (LOH, PC, FRAP, TEAC) were taken at designated time points, represented by blood draw (syringe) icons. Samples taken before and after (randomized, crossover design) 20 minutes CF and TM bouts. ! Treadmil ! PRE 20 minutes of exercise IPE 1Hr 2Hr CF ?CINDY? ! N#! i!h! i!h!k! A B h! h! FIGURE 2. Biomarkers of Oxidative Damage, before and after exercise, Means ? SEM, n=10. A) lipid peroxidation of blood plasma B) Protein Carbonyl blood plasma. * significant from PRE, # significant from IPE, + significant from 1-HP. Black bars = CF, Open bars = TM ! ! N$! ! A B ! * * * * * * FIGURE 3. Biomarkers of Antioxidant Capacity. Means ? SEM, n=10. A) Blood plasma antioxidant potential FRAP B) Blood plasma antioxidant capacity TEAC. * Significantly diferent from PRE. Black bars = CF, Open bars = TM. !