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Delayed muscle soreness, muscle function and evidence of leukocytes in human skeletal muscle following… MacIntyre, Donna Lee 1994

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DELAYED MUSCLE SORENESS, MUSCLE FUNCTION AND EVIDENCE OF LEUKOCYTES IN HUMAN SKELETAL MUSCLE FOLLOWING ECCENTRIC EXERCISE by DONNA LEE MACINTYRE Diploma in Physiotherapy, University of Alberta, 1970 B.S.R.(P.T.), University of British Columbia, 1980 M.P.E., University of British Columbia, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES Interdisciplinary Studies We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1994 © Donna Lee Maclntyre, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  reuL ?-ke.cLL  The University of British Columbia Vancouver. Canada  Date  DE-6 (2/88)  ot 1 .  11  ABSTRACT  The three primary purposes of these studies were to 1) characterize the time course and the relationships among delayed onset muscle soreness, eccentric muscle torque, serum creatine phosphokinase and urinary hydroxyproline in response to two exercise durations; 2) to examine whether there was increased presence of leukocytes in the exercised muscle compared to the contra-lateral non-exercised muscle; and 3) to determine the time course and the relationships among eccentric muscle torque, muscle fatigue, range of motion, the presence of leukocytes, and delayed onset muscle soreness following the eccentric exercise. In the first study, the dependent variables were measured before and after 180 (shorter duration) and 300 (longer duration) eccentric repetitions of the quadriceps muscles. Eccentric torque of the quadriceps muscles was evaluated utilizing the same parameters of range of motion and velocity of movement as the exercise stimulus. Muscle soreness was evaluated by the Descriptor Differential Scale which reflects the sensory (intensity of soreness) and affective (unpleasantness) components of the discomfort. In the second study, a radionuclide technique was used to determine the presence of leukocytes in the exercised muscle. Muscle fatigue was assessed with a power spectrum analysis. The greatest intensity of soreness and unpleasantness was between 20 and 48 hours postexercise. The presence of leukocytes in the exercised muscle was significantly greater than in the contra-lateral non-exercised muscle. Additional data collection before 24 hours post exercise revealed a biphasic response in eccentric torque, not reported previously in humans. Eccentric torque declined immediately after the exercise stimulus and again at 20 to 24 hours post-exercise. Fatigue of the vastus lateralis muscle was most evident at 2 hours post-exercise. The greatest loss of range of motion occurred at 24 hours post exercise. Significant correlations were found between unpleasantness and creatine phosphokinase, intensity of soreness and creatine phosphokinase, and unpleasantness and  111  range of motion. Using a radionuclide imaging technique allowed a quantitative evaluation of the widespread distribution of the leukocytes compared to the smaller sample that would have been evaluated from microscopic examination of human muscle biopsies. Previously only focal injury has been shown due to the sampling limitations of using isolated muscle biopsies.  iv  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  viii  List of Abbreviations  x  Acknowledgements  xii  Dedication  xiii  Chapter One  The Effects of Two Durations of Eccentric Exercise on Delayed Muscle Soreness, Muscle Strength, and Biochemical Markers of Muscle Injury  1  Introduction  1  Purpose  4  Research Hypotheses  4  Methods and Procedures  5  Results  11  Discussion  26  Conclusions  33  References  35  Chapter Two The Effects of Eccentric Exercise on Delayed Muscle Soreness, Muscle Strength and Fatigue, Joint Range of Motion, and the Presence of Leukocytes in Exercised Muscle  40  Introduction  40  Purpose  43  Research Hypotheses  43  Methods and Procedures  44  Results  51  V  Chapter Three  Discussion  75  Conclusions  84  References  86  Concluding Remarks  91 95  References Appendix One  96  Review of the Literature  104  References Appendix Two  Appendix Three  Descriptor Differential Scale  110  Instructions to the Subjects  110  Calculations  110  Reliability and Validity  119  References  123  Power Spectrum Analysis  124  Calculations  124  Reliability and Validity  131  Fatigue of the Rectus Femoris Muscle  134  Fatigue of the Vastus Lateralis and Vastus  Appendix Four  Medialis Muscles  141  References  144  Radio-isotope Investigation of Acute Inflammation  146  Labelling Procedure  146  Analysis of the Presence of Tc-99m WBC  147  Possible Mechanisms Contributing to the  Appendix Five  Responses of Two Subjects  159  References  178  Individual Subject Data Chapter One  180  Individual Subject Data Chapter Two  199  -  -  vi  LIST OF TABLES  1. The Effects of Eccentric Exercise on Muscle Soreness, Muscle Strength and Biochemical Markers  12  2. Two Durations of Eccentric Exercise: Manova Tables  13  3. Two Durations of Eccentric Exercise: Post Hoc Contrasts  20  4. Relationships Among Muscle Soreness, Muscle Strength and Biochemical Markers at Each Test Time  21  5. Indirect Measures of Exercise-Induced Muscle Injury Before and After Eccentric Exercise  .  53  6. The Presence of White Blood Cells in Four Regions of Interest in the Exercised Muscle After Eccentric Exercise 7. Manova Tables for the Indirect Measures  54 55  8. Manova Tables for the Presence of White Blood Cells in Four Regions of Interest 9. Post Hoc Contrasts of the Indirect Measures  56 57  10. The Presence of White Blood Cells in the Four Regions of Interest: Post Hoc Contrasts  71  11. Relationships Among Muscle Soreness, Muscle Strength and Fatigue, and Range of Motion at Each Test Time  72  12. Pearson Product-Moment Correlations for the Descriptor Differential Scale  121  13. Power Spectrum Analysis: Pearson Product-Moment Correlation Coefficients 133 14. Power Spectrum Analysis: Descriptive Statistics of the Median Frequencies of the Quadriceps Muscles 15. Power Spectrum Analysis: Paired T-Tests  138 143  vii  16. Mean Count/Pixel for Each Region of Interest for Each Subject at Each TestTime  151  17. Coefficient of Variation for the Regions of Interest  154  18. Subtraction of the Background  157  19. Technetium-99m White Blood Cells for Each Region of Interest at Each Test Time 20. Normalized Data for Each Region of Interest  166 168  viii  LIST OF FIGURES  1. Muscle Soreness Before and After Two Durations of Eccentric Exercise  14  2. Eccentric Torque Before and After Two Durations of Eccentric Exercise  16  3. Biochemical Markers Before and After Two Durations of Eccentric Exercise  18  4. Regions of Interest for Radio-Isotope Scanning  49  5. Characteristics of Eccentric Exercise: Muscle Soreness  60  6. Characteristics of Eccentric Exercise: Eccentric Torque  62  7. Characteristics of Eccentric Exercise: Range of Motion of the Knee  64  8. The Presence of White Blood Cells in the Four Regions of Interest in the Exercised Muscle 9. Slopes of the Median Frequencies of Two of the Quadriceps Muscles  66 68  10. The Relationship Between Eccentric Torque and Vastus Lateralis Median Frequency at Two Hours Post-Exercise  81  11. Amount of Sensation  111  12. Amount of Unpleasantness  113  13. Subject 1: Intensity of Soreness at 24 Hours Post-Exercise  115  14. Subject 1: Unpleasantness Response at 96 Hours Post-Exercise  117  15. Force and Raw Electromyography (EMG) Signals  125  16. Typical Surface-EMG Power Spectrum  127  17. Regression Lines for the Frequency Spectra  129  18. Power Spectrum Analysis: Rectus Femoris Median Frequency  135  19. Slopes of the Median Frequencies of the Superficial Quadriceps Muscles  139  20. Subject 4: Antero-Distal Region of Interest  148  ix  The Presence of Technetium-99m White Blood Cells in the Four Regions of Interest for Each Subject 21. Subject 1 and Subject 2  161  22. Subject 3 and Subject 4  162  23. Subject 5 and Subject 6  163  24. Subject 7 and Subject 8  164  25. Subject 9 and Subject 10  165  The Responses of Two Subjects Compared to the Group Mean ± One Standard Deviation 26. A. Range of Motion of the Right Knee B. Eccentric Torque  173  27. A. Intensity of Soreness B. Unpleasantness  174  28. A. Vastus Lateralis Median Frequency B. Vastus Medialis Median Frequency  175  x  LIST OF ABBREVIATIONS  ADP  adenosine diphosphate  ATP  adenosine triphosphate  Ca  calcium ion  CMRR  custom mode rejection ratio  CPK  creatine phosphokinase  Cr  creatimne  CV  coefficient of variation  DDS  Descriptor Differential Scale  DOMS  delayed onset muscle soreness  ECM  extracellular matrix  EMG  electromyography  T-IIvIPAO  (RR,SS)-4,8 diaza-3 ,6,6,9-tetramethyl undecane 2, 10-dione bisoxime  ICC  intraclass correlation coefficient  LRP  leukocyte rich plasma  MANOVA  multivariate analysis of variance  02  oxygen  OHP  hydroxyproline  PAF  platelet activating factor  PPP  platelet poor plasma  RF  rectus femoris  RM  repetition maximum  ROT  regions of interest  ROM  range of motion  SD  standard deviation  xi  Tc-99m  technetium  Tc-PYP  technetium pyrophosphate  TNF  tumor necrosis factor  VAS  visual analogue scale  VL  vastus lateralis  VM  vastus medialis  WBC  white blood cells  xii  ACKNOWLEDGEMENTS  I would like to extend my sincere appreciation to Dr. D.C. McKenzie, who was my supervisor throughout the course of my PhD program. I am very grateful to him for his wise counsel; his advice, support, and encouragement in helping me to complete my PhD. I am also grateful to my committee members Dr. A.N. Belcastro, Dr. B.H. Bressler, Dr. -  P.W. Hochachka, Dr. D.M. Lyster, and Dr. W.D. Reid. They each provided me with  ideas and direction, and were generous in sharing their expertise.  I would also like to thank Dr. Jonathan Berkowitz for advising me on the statistical analysis of my data. Dr. Michael Slawnych developed the computer program for the power spectrum analysis. My thanks to him for his assistance and his advice regarding the electromyography. I am also grateful to the staff of the Nuclear Medicine Departments at Vancouver Hospital and at the University Site for their unending patience with me and their cooperation in scheduling my subjects. My thanks also to the staff in the Clinical Laboratory at University Site and the Mineral Metabolism Laboratory at Vancouver Hospital. I am grateful to Mr. Shaffiq Rahemtulla who provided technical assistance with photographs and slides. I also wish to thank Ms. Diana Jesperson, Mr. Randy Celebrini and Mr. Greg McGann for their assistance in collecting the data.  I am indebted to British Columbia Health Research Foundation and Canadian Fitness and Lifestyle Research Institute for their financial support of the studies that comprised my thesis.  xiii  DEDICATION  This thesis is dedicated to  -  my Mother, who taught me what is important in life; both of my parents, who have surrounded their children and grandchildren with unconditional love and support for whatever they have chosen to do; Roger, without whom I would not have completed my PhD. Thank you for the adventures we’ve shared; my friends and colleagues, who in small ways and in substantial ways, helped me along the way. My thanks to all of you.  1  CHAPTER ONE THE EFFECTS OF TWO DURATIONS OF ECCENTRIC EXERCISE ON DELAYED MUSCLE SORENESS, MUSCLE STRENGTH, AND BIOCHEMICAL MARKERS OF MUSCLE INJURY  Introduction  Delayed onset muscle soreness (DOMS) is a sensation of discomfort associated with movement or palpation usually felt in skeletal muscle 24 to 72 hours following unaccustomed muscular exertion (1). The sensation of discomfort following overuse of a muscle can be very severe and appears to have characteristics similar to pain. Certain investigators have suggested that there is both a sensory and an affective component to pain (2, 3). The sensory component refers to the physiological stimulation of the peripheral receptors and nerves but this sensory component is further modulated by the individual’s past experience where attitudes and psychological variables may influence description of the sensation (4). By attending only to the sensory aspect of pain, the complete picture of the pain process is overlooked (5). As muscle soreness is a sensation of discomfort which may be associated with pain, it would seem appropriate to assess both the intensity of muscle soreness (sensory component) and the related unpleasantness (affective component) associated with the sensation.  Although discomfort may have more than one component, studies performed to date examining DOMS have reported assessment of the intensity of the sensation but consideration of any affective component of DOMS has been neglected (6-10). Further, investigations examining DOMS have often used a visual analog scale (VAS) (7) or a rating scale where a given number was chosen based on the corresponding adjective (6,  2  9-11). When used for repeat testing, these scales may suffer from scaling error, which includes repeatedly using the same category or part of the line, or remembering a past specific response (2). This may limit the usefulness of the data derived, especially in extrapolation of the results to different populations. The Descriptor Differential Scale (DDS) enables collection of multiple responses and minimizes the scaling error.  Although it is clear that DOMS results from overuse of muscle, especially as a result of eccentric exercise, the specific etiology is not well understood. In 1990, Stauber and colleagues (12) suggested that DOMS was due to a complex set of reactions involving disruption of connective tissue and the muscle fibre. Similar to Stauber and colleagues, Smith (13) reported that mechanical disruption to the muscle fibre and connective tissue is a result of the unaccustomed eccentric exercise, but the author also suggested that, as a result of the muscle injury, an acute inflammatory response begins. Similarities between DOMS and acute inflammation include pain, swelling and loss of function as measured by muscle strength.  Creatine phosphokinase (CPK) has been shown to increase after eccentric exercise but it does not have the same time course as the perception of soreness (12, 14). The different time course may be related to the delay of the intracellular proteins entering the blood (12, 14), or the divergent response may be due to the location of the pain fibres in the connective tissue and not in the muscle fibre (15).  An early investigation by Abraham examined a marker of connective tissue damage, the appearance of urine hydroxyproline (OHP), which was reported to be closely related to the time course of DOMS (6).  Abraham (6) also recommended that  3  standardizing the OHP excreted by the creatimne (Cr) excreted over 24 hours should give a more sensitive measure of the changes in Ol-IP.  Studies which have related muscle force to DOMS have failed to find a close association in the time course of these two parameters. However, in previous studies a different protocol was used to assess force compared to the protocol used to induce the muscle injury.  Newham and colleagues (16) used maximum voluntary isometric  contractions of the knee extensors to evaluate strength but utilized a bench stepping exercise as the stimulus to induce DOMS. Fnden and colleagues (17) used isokinetic (at angular velocities of 180 and 300 degrees per second) and isometric strength to assess performance following an eccentric bicycle exercise to elicit muscle soreness. In the above mentioned studies, it is quite possible that different populations of muscle fibres contributed to the performance tests compared to those affected by the initial bout of exercise used to stimulate DOMS. A performance test more similar to the intervention used to induce the original muscle damage or muscle soreness would be a better choice to assess functional outcome.  Tiidus and lanuzzo (18) have reported a lack of quantification regarding the amount of exercise required to produce DOMS or an increase in the activity of CPK. Subjects in their study performed concentric and eccentric knee extensor contractions under three different exercise regimes intensity, duration and constant total work. Their -  results indicated that as exercise intensity increased (from 35% of 10 repetitions maximum [RM] to 90% of 10 RM through 150 repetitions) the sensation of muscle soreness increased. As well, long duration exercise (300 repetitions vs 100 or 200) produced elevated CPK levels and greater muscle soreness. More recently Newham and colleagues (19) have reported that muscle soreness is greater when the muscle is exercised at a longer length compared to a shorter length.  4  In this study, both the sensory and affective components of DOMS were assessed by using the Descriptor Differential Scale (DDS). For each assessment of DOMS, the subjects scored their intensity of soreness and unpleasantness immediately following the test of eccentric torque. This standardized the experience for the subject’s assessment of DOMS. In addition, the same parameters of exercise (position, range of motion, velocity of motion) were used to evaluate eccentric torque on subsequent days, as the eccentric exercise stimulus used to induce DOMS. Therefore the performance tests were likely recruiting a similar motoneuronal pool as the muscle fibres affected by DOMS. And finally, several parameters (DOMS, eccentric torque, CPK and OHP/Cr) were measured in the same study.  Purpose  The purpose of this study was to examine the time course and the relationships over time among DOMS, muscle torque and indicators of muscle damage (CPK and OHP/Cr) in a sedentary group of individuals in response to two levels of eccentric exercise duration.  Research Hypotheses  1. The magnitude of responses following the exercise stimulus will be directly related to the duration of the exercise. 2. The greatest perception of DOMS (measured as intensity of soreness and unpleasantness) will occur between 24 and 48 hours following the exercise stimulus, at the same time as the greatest OHP/Cr response, but earlier than the greatest response in CPK.  5  3. The timing of the greatest loss of eccentric torque will be different from the greatest perception of DOMS.  Methods and Procedures  Subjects  Twenty individuals (16 females and 4 males) between the ages of 19 and 52 were recruited into the study. However, the data from one subject was not included in the analysis as she participated in another eccentric exercise activity (water skiing) during the same week as data collection for this study. Individuals were excluded from the study if they engaged in recreational exercise of more than 4 hours/week, were running, jogging or lifting weights for the lower extremities, or involved in competitive sport. In addition, those individuals who may have had cardiovascular, neurological or musculoskeletal conditions that may have compromised their ability to perform the testing procedures or which may have posed a hazard to the individual as identified by their physician (ie.  -  cardiac disease, a chronic neurological disease, degenerative knee joint disease, a recent soft tissue injury of the lower extremity) were excluded. Approval for this study was granted by the Clinical Screening Committee for Research Involving Human Subjects at the University of British Columbia.  Protocol  The protocol was a randomized cross-over design, such that each of the subjects performed two different durations (shorter and longer) of the exercise stimulus in order to  6  induce DOMS. Following informed consent, subjects were randomly assigned to the order of the exercise stimulus (shorter first or longer first). Subjects completed baseline measures (pre-test) of the DDS describing DOMS (intensity of soreness and unpleasantness), eccentric torque of the quadriceps, serum levels of CPK, and urine levels of Ol-IP and Cr. This was followed by repetitive fatiguing eccentric contractions of the quadriceps at either shorter or longer duration (the exercise stimulus) on an isokinetic dynamometer.  The DDS, tests of muscle torque, and blood and urine samples were  repeated 24 hours (day 1), 48 hours (day 2), 96 hours (day 4), and 168 hours (day 7) after the exercise session. At least twelve weeks after the first testing session the protocol was repeated on the same leg in the same subject at the other duration. This interval was chosen because Evans found that repair of damaged muscle may take as long as twelve weeks (20).  Methods  Descriptor Differential Scale  The intensity of soreness and unpleasantness of muscle soreness was assessed using the DDS (2). This scale contains 12 descriptor items for each dimension assessed, and for each item, the subject indicated if the intensity of soreness and then the unpleasantness was either equal in magnitude to that implied by each anchoring descriptor or how much greater or lesser on a 21-point graphic scale (Appendix Two). For all subsequent assessments, subjects completed the scales immediately after a short session (the eccentric torque test) of a similar exercise that was used to initiate DOMS. To assess intensity of soreness, the subjects were instructed to evaluate DOMS elicited throughout the quadriceps muscles during the eccentric torque test.  To assess  7  unpleasantness, they were asked to evaluate DOMS elicited throughout the quadriceps during their daily activities.  Gracely and Kwilosz (2) have reported the DDS to be reliable in the assessment of pain between hours one and two (Pearson product-moment correlations of 0.82 for sensory intensity and 0.78 for unpleasantness) in a group of dental patients.  See  Appendix Two for reliability and validity of the DDS in subjects with DOMS.  Eccentric/Concentric Torque of the Quadriceps Muscles  The subjects were seated on the KinCom (Medex Diagnostics of Canada, Coquitlam, B.C.) isokinetic dynamometer, with their hips at 80 degrees, their back supported and the pelvis and thigh stabilized on the bench. The centre of rotation of the KinCom was positioned opposite the centre of the knee joint line. The resistance pad was positioned distally against the tibia so that the lower edge of the pad was at a point on the lower leg that was 75% of the length of the fibula. The angular velocity was set at 30 degrees/second through a range of 60 degrees at a long muscle length (110 50 degrees -  of knee flexion).  The subjects performed three submaximal (the subjects were instructed to push against the pad at approximately 50% of their maximal effort) and one maximal (the subjects were instructed to push against the pad as hard as they could) practice contractions, followed by four maximal contractions. A two minute rest was interposed between the warm-up contractions and the four maximal repetitions. The data was collected during the last four maximal contractions, saved onto disk and subsequently analyzed as the average eccentric torque of the knee extensors over repetitions two to four.  8  Farrell and Richards (21) have reported the measurements of the KinCom system to be repeatable (repeated loading and unloading of a strain gauge) and accurate to known weights (Intraclass Correlation Coefficient [I.C.C.]  =  0.99) in static testing. During  dynamic testing, the applied force from trial to trial resulted in an I.C.C. of 0.95. Reliability of concentric and eccentric torque measurements (I.C.C.) on the KinCom have been reported to range from 0.93-0.98 for both slow (30 degrees/second) and fast speeds (180 degrees/second) in groups of healthy active subjects (22,23).  The Exercise Stimulus  Subjects were seated on the KinCom as described above and performed repeated eccentric contractions (110-50 degrees of flexion) at a slow speed (30 degrees/second) using the passive mode of the machine. The subjects were instructed to lift their leg during the concentric movement of the lever arm but not to push against the lever arm so that production of lactic acid would be limited (14).  When the machine changed  direction they were instructed to resist the eccentric movement of the lever arm maximally throughout the range of motion. Subjects had continuous visual feedback of their force from the computer screen. In the shorter exercise stimulus the subjects completed 180 repetitions  -  18 sets of 10 repetitions of maximal eccentric contractions/set  with a one minute rest between each set. For the longer exercise stimulus subjects completed 300 maximal eccentric contractions  -  30 sets of 10 repetitions with each set  beginning every minute for 30 minutes. This allowed a 20 second rest between each set.  9  Analysis of Creatine Phosphokinase  Venipuncture was performed at the time intervals noted in the protocol. The blood samples were immediately taken to the laboratory where they were centrifuged. Eleven microlitres (pL) of the specimen were placed on a Kodak Ektachem clinical chemistry slide (Eastman Kodak Co., ROchester, N.Y.). This analysis uses creatine phosphate and adenosine diphosphate as substrates to generate creatine adenosine triphosphate. In a coupled reaction sequence, hydrogen peroxide (which is produced in stoichiometric equivalents to ATP in the initial reaction) oxidizes a dye precursor. The rate of chromophore production is monitored by reflectance spectrophotometry at 670 nm and used to measure CPK activity.  Analysis of Creatinine  Ten mL of the 24 hour urine collection were sampled and taken to the laboratory for analysis. In the laboratory, 10 j%L of the urine specimen were deposited on a Kodak Ektachem clinical chemistry slide.  Creatinine diffused to gel layers where it was  hydrolyzed to creatine. During the initial reaction phase, endogenous creatine was oxidized by reagents in the slide. Creatine was converted to sarcosine and urea by creatine amidinohydrolase. The sarcosine was oxidized to glycine, formaldehyde and hydrogen peroxide. Rate determination was made at 3.85 and five minutes. The rate of change between the two readings was proportional to the creatimne concentration in the sample.  10  Analysis of Hydroxyproline  Subjects were informed of the importance of a diet low in gelatin content in order to minimize the influence of endogenous sources of Ol-IP. Urine samples were collected over 24 hours and refrigerated during the collection period. After thorough mixing, one 8 mL aliquot was sampled and stored at -70 degrees centrigrade until the analysis was performed.  Urine was assayed using the technique developed by Hughes and co-workers (24). In this method, OHP is derivatized with 4-chloro-7-nitrobenzofurazan, with subsequent estimation by reversed phase “high performance” liquid chromatography.  Statistical Analysis  A two-way multivariate analysis of variance (MANOVA) for grouping factors (exercise duration) and repeated measures over time (24, 48, 96, 168 hours) was used on each of the dependent variables (DDS intensity of soreness score, DDS unpleasantness score, eccentric torque, CPK, OHP/Cr) to examine for differences over time and between exercise durations. Post hoc analyses examined contrasts at each test time compared to the pre-test and between test times. Only those variables that were significant over time were examined as the multivariate F test is more robust than the univariate post hoc tests (25). Correlation analyses (Pearson product-moment correlation) were used to examine the relationships among DOMS, eccentric torque of the quadriceps, CPK, and OHP!Cr. The significance levels were set at p < 0.05.  11  Results  There were no significant interactions and no significant differences between exercise durations (180 repetitions vs 300 repetitions) for any of the dependent variables (Table 2). However, intensity of soreness, unpleasantness and eccentric torque were all signficantly different over time (p<0.001). The greatest intensity of soreness was at 24 to 48 hours post-exercise (Figure 1A), while the greatest unpleasantness was at 48 hours post-exercise (Figure 1B). The greatest loss of eccentric torque was at 24 hours postexercise (Figure 2). There were no significant differences between exercise durations or over time for either CPK or OHP (Figure 3). The means and standard deviations (SD) for each of the dependent variables for both exercise durations over time are presented in Table 1.  Table 3 summarizes the post hoc contrasts for the dependent variables. Intensity of soreness (Figure 1A) and unpleasantness responses (Figure 1B) were all significantly higher than the pre-test, except for the final testing time at 168 hours (7 days) when both the intensity of soreness and unpleasantness responses had returned to baseline levels. Between test time periods, only the intensity of soreness responses at 24 and 48 hours were not significantly different. Eccentric torque at 24 and 48 hours were significantly lower than the pre-test (Figure 2). When contrasting the eccentric torque responses between testing times only the 24 and 48 hour responses were not significantly different, similar to the analysis of the intensity of soreness.  Correlation matrices (Table 4) revealed that the unpleasantness score and the CPK response, at 168 hours (7 days) after the shorter exercise duration, had the highest correlation at 0.90 (rZ=0.81). Other significant correlations (p<0.05) ranged from 0.51  -  0.76 between CPK and intensity of soreness from 96 hours to 168 hours after the shorter  62.4 ± 1.3 156.4 +77.1  CPK (lU/l)  Data are means  163 ±103.2 8660.2 ±3324.0 17.4 ±7.0 16.2±7.4  104.9±44.6 1321.9 ±4106.5  3.5±8.4 -3.3 ±6.6  16.3 ±53  138.9 ±81.7 8384.3 ±4462.6  216.4 ±253.2  109 ±41.6  -4.4 ±5.3  2.9 ±8.5  96 Hours  136.7±84.8 8532.6 ±4592.9  496.7 ±1077.3  3.3 ±6.6 89.7  8.9 ±6.3  8941.0 ±79.4 17.4 ±9.0  181.3 ±134.8 162.9 ±140.7  87.5 ±30.1  10.1 ±5.6 2.1 ±•  48 Hours  15.3 ±8.7  365.6 ±670.8 132.9 ±• 1 8350.2 ±3627.3  -9.5 ±0.8 120.8  -8.5 ±1.8  143.1 ±87.9 9233.6 ±483 1.8 15.7 ±3  125.4 ±48.4 397.8 ±686.9  -8.5 ±2.8  -6.2 ±6.5  168 Hours  SD for 19 subjects. *Creatine Phosphokinase, t=Hydroxyproline, =Hydroxyproline/Creatimne  Creatimne(umol/l) OHP/Cr  155.4 ±107.8  335.2  87.9  9.6 ±4.3 -0.5 ±4  8870.0 ±3523.5 10222.1 ±6042.8 18.7 ±7.6 14.2 ±4.6  114.4 ±• 1  Torque (N.m)  OHP (mol/l)  -9.7 ±0.9  Unpleasantness  Longer exercise stimulus (300 repetitions) Intensity of Soreness -8.1 ±2.9  OHP/Cr  Ol-IP t (ptmol/l) Creatinine (jtmol/l)  84.9  -0.4 ±6.3  8.4 ±6.0  24 Hours  269.2 ±203.5 183.2 ±142.3 141.4±114.2 7293.9 ± 833.4 10745.6 ±6237.8 18.8 ±• 16.0 ±•  71.4 ±38.9  -9.7 ±0.5 121.7 ±46.3  Unpleasantness  Torque (N.m) CPK* (lU/l)  -7.8 ±4.0  Intensity ofSoreness  Shorter exercise stimulus (180 repetitions)  Pre-test  THE EFFECTS OF ECCENTRIC EXERCISE ON MUSCLE SORENESS, MUSCLE STRENGTH AND BIOCHEMICAL MARKERS  TABLE 1  I)  I-  13  TABLE2 TWO DURATIONS OF ECCENTRIC EXERCISE: MANOVA TABLES  Source (of Variation)  Intensity of Soreness Within Cells Between Durations Within Subjects Time Within Cells Duration xTime Unpleasantness Within Cells Between Durations Within Subjects Time Within Cells Duration xTime Eccentric Torque Within Cells Between Durations Within Subjects Time Within Cells Duration xTime Creatine Phosphokinase Within Cells Between Durations Within Subjects Time Within Cells Duration xTime Hydroxyproline Within Cells BetweenDurations Within Subjects Time Within Cells Duration xTime *  1 o . 0 p<  df  MS  18 1 72 4 72 4  40.91 11.90 27.85 2766.47 19.88 17.33  18 1 72 4 72 4  38.81 2.55 18.38 1096.20 9.73 7.54  18 1 72 4 72 4  617.00 219.00 488.90 10605.00 343.05 196.61  18 1 72 4 72 4  3471457.4 3972740.4 1493968.9 2440390.2 1344252.7 2159078.8  18 1 72 4 72 4  6606.97 1197.52 4908.27 4825.30 5395.84 5332.74  F  0.29 99•34* 0.87 0.07 59.64*  0.78 0.35 21.69* 0.57  1.14 1.63 1.61 0.18 0.98 0.99  14  Figure 1. Muscle Soreness Before and After Two Durations of Eccentric Exercise. Values are means ± SD. There were significant differences over time p<O.OO1. *  Significantly different from the pre-test p<O.OO1.  test to previous test p<O.OO1. A. Intensity of Soreness B. Unpleasantness  On all graphs where there is a pre-test, pre = pre-test.  +  Significantly different this  ci)  0 C  I,,  0  r\)  -U, CD  C  I  U,  .  .  -i  U,  MEAN SCORE  ....  I  -  U,  U)  0 C  0,  0)  HOD  -‘  CD  0  +  F  F  F  F  1  1  0 ri  iI  (i  MEAN SCORE  [± rnH  -S CD  -a.  -I,  C.,,  I-.  16  Figure 2. Eccentric Torque Before and After Two Durations of Eccentric Exercise. Values are means ± SD. There were significant differences over time p<O.OO1. *  Significantly different from the pre-test p<O.OO 1.  test to previous test p<O.O 1.  +  Significantly different this  c  0  z  m  H  cc  cc  CD  -o  0 ‘T  0  -  +  +  ----  OD 0  I  Ni 0  —  _Y  0  -  ECCENTRIC TORQUE (Nm)  —  Ni C 0  F’.)  CD  -s  18  Figure 3. Biochemical Markers Before and After Two Durations of Eccentric Exercise. Values are means ± SD. A. Creatine Phosphokinase (CPK) B. Hydroxyproline C. Creatinine D. The hydroxyproline/creatimne ratio  a  c:  r  <  a C) z  200  250  300  350  0  400  800  1200  1600  2000  •  •  B  A  Pre  +  Pre  24  I F:  24  48  I  48  96 TIME (Hours)  I  96 TIME (Hours)  •  - -  168  LONGER  SHORTEI]  168  Figure 3 C  a  C)  x  0  0  •  D  -  •_......  -  t  -Ic  10-  15  20  25  30  1000  5750  1.050104.  w  1.52510 E  2.000 i0  Pre  4  Pre  24  ....•4•.•  I  24  7,  I  48  I  48  I  I  96 TIME (Hours)  L_....•._  ----SHORTER • LONGER  I  96 TIME (Hours)  LONGER  •  —  168  I I  I  168  I•-•——•—i•-—--•-  SHORTER  --I--  -  cc  I—.  20  TABLE3 TWO DURATIONS OF ECCENTRIC EXERCISE: POST HOC CONTRASTS  Umvariate F  p  Intensity of Soreness df 1,18 pre 24 hr pre-48hr pre-96hr pre-168hr  125.80 313.77 41.60 0.28  <.001* <.001* <.001* 0.60  24hr-48hr 48 hr-96 hr 96hr-168hr  0.62 24.00 60.82  0.44 <.001* <.001*  69.75 111.84 32.47 3.50  <.001* <.001* <.001* 0.07  16.69 52.55 33.64  0.001* <.001* <.001*  32.79 23.59 4.02 1.01  <.001* <.001* 0.06 0.33  0.38 13.70 23.48  0.54 0.002* <.001*  Source  -  Unpleasantness pre-24hr pre-48hr pre-96hr pre-168hr  df 1,18  24hr-48hr 48hr-96hr 96 hr 168 hr -  Eccentric Torque pre-24hr pre-48hr pre -96 hr pre- 168 hr  df 1,18  24hr-48hr 48hr-96hr 96 hr 168 hr -  *signifijnt differences between tests  21  TABLE 4 RELATIONSHIPS AMONG MUSCLE SORENESS, MUSCLE STRENGTH AND BIOCHEMICAL MARKERS AT EACH TEST TIME CORRELATION MATRICES Shorter Exercise Stimulus (180 repetitions) Test Times (hours) Pre-test  24  48  96  168  Intensity of Soreness Test Times (hours) Unpleasantness Pre-test 0.13 24 48 96 168 Pre-test 24 48 96 168 Pre-test 24 48 96 168 Pre-test 24 48 96 168  0.72* 0.54* 0.81* 0.93*  Eccentric Torque 0.18 -0.11 0.30 0.34 -0.18 Creatine Phosphokinase 0.01 0.34 0.39 0.51* 0.76* Hydroxyproline 0.23 -0.04 0.18 0.03 -0.07 Unpleasantness  Eccentric Torque Pre-test -0.42 24 48 96 168  -0.16 0.18 0.19 -0.22  22  Shorter Exercise Stimulus cont. Test Times (hours) Pre-test  24  48  96  168  Unpleasantness cont. Test Times (hours) pretestCatisPh011e 0.35 48 96 168  0.36 0.52* 0.90 *  pretest0xne 0.16 0.16 96 168  -0.06 -0.18 Eccentric Torque 0.14 0.15  96 168  -0.03 -0.14  0.23 0.38 96 168  0.13 -0.09 Creatine Phosphokinase -0.15 0.10  96 168  -0.16 -0.10 Creatine  Pre-test 24  0.30 0.06 0.09  96 168  0.01 -0.10  23  Shorter Exercise Stimulus cont. Test Times (hours) Pre-test  24  48  96  168  Hydroxyproline Test Times (hours) Creatimne Pre-test 24 48 96 168 * =  0.73*  0.74* 0.79* 0.89* 0.57*  p<0.05  Longer Exercise Stimulus (300 repetitions) Test Times (hours) Pre-test  24  48  96  168  Intensity of Soreness Test Times (hours) Unpleasantness 0.67* Pre-test 24 48 96 168 Pre-test 24 48 96 68 Pre-test 24 48 96 168 Pre-test 24 48 96 168  0.63*  0.52* 0.76* 0.51*  Eccentric Torque 0.04 0.15  0.20 0.17 -0.18 Creatine Phosphokinase 0.20 0.29 0.11 0.34 0.58*  Hydroxyproline 0.13 0.12 0.25 0.17 -0.31  24  Longer Exercise Stimulus cont. Test Times (hours) Pre-test  24  48  96  168  Unpleasantness Test Times (hours) Eccentric Torque -0.18 Pre-test 24 48 96 168 Pre-test 24 48 96 168 Pre-test 24 48 96 168  -0.08 -0.04 -0.19 -0.08  Creatine Phosphokinase 0.05 0.23 0.39 0.10 -0.10 Hydroxyproline -0.06 -0.02 0.17 0.42 -0.16 Eccentric Torque  Creatine Phosphokinase Pre-test 0.45 24 0.21 48 96 168 Pre-test 24 48 96 168  0.18 0.01 0.04  Hydroxyproline 0.02 0.26 0.13 -0.04 -0.01 Creatine Phosphokinase  Hydroxyproline Pre-test -0.06 24 48 96 168  -0.30 -0.04 -0.08 -0.20  25  Longer Exercise Stimulus cont. Test Times (hours) Pre-test  24  48  96  168  Creatine Phosphokinase cont. Test Times (hours) Creatinine 0.43 Pre-test -0.15 0.27  48  0.01 0.04  168 Hydroxyproline Creatinine 0.57* Pre-test 0.83* 0.60*  0.85*  96 168 * =  0.65* p<0.05  26  exercise stimulus, and 0.58 at 168 hours after the longer exercise stimulus. Regarding CPK and unpleasantness, significant correlations ranged from 0.52 0.90 at 96 and 168 -  hours after the shorter exercise stimulus. Other significant correlations were found between the intensity of soreness and unpleasantness from 24 hours post-exercise to 168 hours post-exercise for the shorter exercise stimulus, in which the correlations ranged from 0.54 to 0.93. The correlations between intensity of soreness and unpleansantness after the longer exercise ranged from 0.51 to 0.76 and were significant at all test times. There were also significant correlations between OHP and Cr at all test times over both exercise durations. There were no other significant correlations among the dependent variables.  Discussion  Because Tiidus and lanuzzo (18) found that intensity and duration of exercise affect both post-exercise serum enzyme activities and delayed muscle soreness, one of the aims of this study was to determine if there was a difference in outcome measures between two durations of exercise. Even though the number of repetitions of the shorter exercise stimulus (180) was 60% that of the longer exercise stimulus (300 repetitions), and the rest periods were longer between sets during the shorter exercise, the outcome  measures were not significantly different between the two exercise durations. In fact they were essentially the same for all of the dependent variables except CPK at 48 and 96 hours. But despite the large differences between the exercise durations for CPK, the extreme variability between subjects eliminated the possibility of finding significant differences (Table 1, Figure 3).  27  Tiidus and lanuzzo (18) reported their highest CPK response at 24 hours; before the highest soreness response at 48 hours. In this study the highest CPK responses were after the highest soreness responses. However, there were some differences between the protocols of this study and that of Tiidus and lanuzzo. In this study the resisted quadriceps exercise was eccentric only, while Tiidus and lanuzzo utilized a concentric and eccentric protocol. Their subjects worked at 70% of 10 RM but in this study the subjects were encouraged to give their best effort throughout the exercise. However, because of visual feedback of the force during the exercise stimulus, it was noted that the force decreased as the exercise progressed in this study. Clarkson and colleagues (10) have proposed that different exercise regimes may result in different mechanisms producing muscle soreness.  All of the intensity of soreness responses from 24 hours to 96 hours were significantly higher than the pre-test with the greatest soreness at 48 hours after the shorter exercise and at 24 hours after the longer exercise (Figure 1A). By 168 hour (7 days) the intensity of soreness had returned to the baseline. The perception of unpleasantness was greatest at 48 hours after both exercise durations (Figure 1B). The responses at all testing times were significantly higher from the pre-test except the response at 168 hours (7 days) when the perception of unpleasantness had returned to baseline. Sore muscles are often described as stiff, aching or tender (1, 26); sensations that are not only associated with intensity of discomfort. Individuals may also have an affective, or emotional, response to the soreness. In assessing the sensation of pain, Meizack (5) has suggested that by only attending to one dimension the complete picture is overlooked. The DDS is one scale that measures both the sensory and affective components of sensation.  28  However, the results of this study suggest that either unpleasantness (the affective dimension of sensation) was not a prominent experience for these subjects or that the DDS differentiated between the two domains of discomfort. The highest unpleasantness mean was 3.3 while the highest intensity of soreness mean was 10.1, indicating that overall the perception of unpleasantness was less than the perception of the intensity of soreness (Figure 1). Because muscle soreness after exercise is such a common experience, the subjects may have been less bothered by the unpleasantness of the discomfort knowing that it would soon subside. Further study might compare the intensity of soreness and unpleasantness responses of healthy subjects with DOMS to individuals who are beginning exercise after a lengthy immobilization and whose response to a novel exercise has not been extensively reported. It may be that duration or intensity of soreness that is not predictable might have a greater affective component. Or, it could be that the affective dimension is not a prominent experience in muscle soreness compared to pain syndromes.  The greatest decline in eccentric torque was at 24 hours after both exercise intensities, although responses at 24 and 48 hours were both significantly lower than the pre-test (Figure 2). Other investigators have reported that the greatest decline in muscle strength is immediately after the exercise stimulus followed by a slow recovery for a week or longer (10, 27). In this study, eccentric torque data was not collected immediately after the fatiguing exercise but it did take 96 hours (4 days) for torque to return to pre-exercise levels (Figure 2).  It is interesting to note that intensity of soreness was highest and eccentric torque was lowest between 24 and 48 hours. Figures 1A and 2 illustrate an extended time period over which the greatest responses occurred. However, similar to other reports in the literature there were no significant correlations between eccentric torque and the intensity  29  of muscle soreness (28). As suggested by Faulkner and colleagues (29), it may be that there is more than one mechanism contributing to the events in muscle after strenuous eccentric exercise. Friden and colleagues (17) have reported Z-line broadening and streaming in the muscles of human subjects immediately after eccentric exercise. This damage to the muscle may explain the loss of muscle strength that has been observed immediately following eccentric exercise. Smith (13) has suggested that the sensation of muscle soreness evident between 24 and 48 hours post-exercise may be associated with an acute inflammatory response. Thus, first a mechanical injury and then a biochemical response may explain the functional responses of the muscle after eccentric exercise.  Nosaka and colleagues (30) have reported that eccentric exercise produces the largest increase in CPK and a different time course from other types of exercise. Typically CPK does not begin to increase until 24-48 hours after eccentric exercise and reaches peak values three to six days after the exercise (30). Once again, mode of exercise could explain the differences in the results of this study and those of Tiidus and lanuzzo (18) who observed a peak CPK response at 24 hours with a concentric/eccentric exercise routine. In this study, the CPK response after the shorter exercise stimulus appeared biphasic with the first increase at 24 hours and the greatest response at 168 hours (7 days). After the longer exercise stimulus, CPK increased up to 96 hours (4 days) and then decreased at 168 hours (7 days)[Figure 3A]. Nosaka and colleagues (30), have suggested that the delay in the rise of CPK may be due to a slowing of the transport of enzymes through the lymph due to swelling and connective tissue damage. Clarkson and Tremblay (31) proposed that the post-eccentric exercise appearance of CPK in the blood is an indication of the onset of necrosis. At present there is no definitive explanation for this long delay.  30  The characteristic intersubject variability of the CPK response after exercise has been observed by others (10,32). Clarkson and colleagues (10) have suggested three groupings of CPK response. High responders are those individuals with a peak CPK response over 2000 lU/L; medium responders have a peak between 500 and 2000 lU/L and low responders are those with a peak CPK response of less than 500 lU/L. Clarkson and colleagues (10) also compared the CPK responses in each of the groups to other outcome measures such as muscle soreness, isometric strength and joint angle. They concluded that low CPK responders have smaller changes in the other measures as well. In this study there were not enough subjects to analyze the outcome measures according to these groupings. However, the CPK data was examined for extreme scores and analyzed without the data from the three subjects who had peak CPK responses above 2000 lU/L. The pattern of the responses over time was still the same as the full data set. Most importantly the pattern of responses was similar to what has been reported in the literature (30). The greatest responses in CPK were observed from four to seven days post-eccentric exercise.  Similar to CPK, the responses of OHP after strenuous exercise (Figure 3B) were not significantly different between durations or over time. In attempting to determine the mechanism underlying delayed soreness, Abraham first reported upon the presence of OHP after a step-exercise (6). He suggested that changes in connective tissue metabolism could be observed by monitoring changes in OHP excretion. Elevated urinary excretion of OHP following exercise may be interpreted as increased catabolism of collagen, a major constituent of connective tissue (15,33). Abraham (6) recommended that OHP be reported relative to Cr because 90% of OHP is rapidly metabolized. In addition, the kidneys efficiently reabsorb the OHP excreting only approximately 10% into the urine. Because Cr excretion may be a reflection of kidney filtration, standardizing the Ol-IP  31  excreted relative to Cr excreted over 24 hours would give a more sensitive measure of changes in OHP metabolism.  Even though Abraham found no significant increase in OHP or OHP/Cr, he found a significant correlation between OHP/Cr and soreness (6). From these results, he proposed that exercise induced muscle soreness may be related to disruption of the connective tissue in the muscle.  Stauber (34) suggested that the OHPICr ratio is only useful to standardize for kidney filtration under normal conditions, but in abnormal situations such as after kidney damage, pregnancy, and muscle injury the relationship between Ol-IP and Cr changes substantially. Considerable muscle damage can cause Cr release from muscle and elevated Cr levels in the urine. Therefore, in this situation OHP and Cr data should be examined separately. Figure 3B and 3C illustrates the OHP data and Cr data respectively. After the shorter exercise stimulus, the pattern of the Cr response was very similar to the shorter exercise OHP response. In fact, significant correlations were found between OHP and Cr at all testing times (Table 4) supporting Stauber’s hypothesis that Cr is released from damaged muscle. After the longer exercise stimulus Cr was highest at 24 hours, a response which was not similar to OHP or CPK both of which were high at 96 hours. However, significant correlations still existed at all testing times between OHP and Cr (Table 4). There were no significant correlations between CPK and Cr (Table 4). Figure 3D illustrates the OHP/Cr ratio. Just as Stauber had predicted, the Cr levels rose to a greater extent than OHP because the OHP/Cr ratio declined at 24 hours and even though it rose after that it never recovered to baseline levels. In other words, the denominator (Cr) increased to a greater extent than the numerator (OHP) causing a decrease in the value of the ratio compared to the baseline measure.  32  Considering the difficulties in the measurement of OHP in this study, as well as the results of Seaman and lanuzzo (35) and Horswill and colleagues (36), there is lack of support for Abraham’s conclusion that DOMS is related to connective tissue damage in muscle (6). However, differences in exercise protocols and differences in sampling procedures in all of the studies are two major reasons why the results may differ and why it is difficult to compare results. Further study is needed in this area, particularly studies with similar protocols. Dietary restrictions are also an important consideration in OHP measurements. Subjects should abstain from foods high in collagen (meats, fish, poultry and foods containing gelatin) as urinary OHP is sensitive to dietary intake (37). In addition, the OHP urinalysis procedures outlined by Hughes and colleagues (24) required a 24 hour urine collection. These criteria required a high degree of subject compliance, and non-compliance had the potential to contribute considerably to the variability. From the results of this study, the fact that the standard deviation was high at the pre-test suggests that diet and subject compliance may have been a factor contributing to the variability of the OHP measure.  There were significant correlations between the intensity of soreness and CPK, and between unpleasantness and CPK at the last two testing times (Table 4). The highest correlation (0.90) was between unpleasantness and CPK at 168 hours (7 days) after the shorter exercise stimulus. Even though the group responses had essentially returned to baseline, those subjects with the highest CPK responses had the highest unpleasantness scores.  Rodenburg and colleagues (28) also have reported significant within-measure correlations and between-measure correlations for 27 subjects for DOMS and CPK at five time periods over 96 hours following eccentric exercise of the elbow flexors. Correlations among DOMS and CPK ranged from 0.36 to 0.58 from 48 to 96 hours post-  33  exercise. In this study signficant correlations were found between intensity of soreness and CPK from 96 to 168 hours post-exercise (0.51 0.76) and between unpleasantness -  and CPK over the same time period (0.52 0.90) (Table 4). The highest correlations in -  this study were at 168 hours post-exercise. Even though the group means were returning to baseline for these dependent variables, the high correlations suggest that there was a relationship between DOMS and CPK from four to seven days post-exercise.  Conclusions  From the results of this study, there were no significant differences between the two exercise durations for any of the outcome measures. The primary difference in the protocol between the two levels of exercise was in regard to duration, or number of repetitions, and although the shorter exercise duration (180 repetitions) was 60% that of the longer duration (300 repetitions) the outcome responses were essentially the same. This differs from the results of Tiidus and lanuzzo (18) but their exercise protocol was concentric and eccentric knee extension while the protocol in this study was eccentric knee extension only.  The functional outcome measures (intensity of soreness, unpleasantness and eccentric torque) were all significant over time (p<0.001). The greatest perception of the intensity of soreness was between 24 and 48 hours for both exercise durations. The greatest perception of unpleasantness was at 48 hours post-exercise for both exercise durations. The greatest loss of eccentric torque occurred at 24 hours post-exercise,  34  however, there were no significant correlations among eccentric torque and the other outcome measures.  In this study, a sensory descriptor scale (DDS) that assessed more than one dimension of discomfort was utilized. Using the DDS, the subjects’ perception of intensity of soreness as well as their perception of unpleasantness of soreness the -  affective or emotional response to discomfort was examined. The results of this study -  suggest that the affective domain was not a primary experience for the subjects, in that the highest unpleasantness response was much lower than that of the intensity of soreness.  There were no significant differences between exercise durations or over time for the biochemical outcome measures (CPK and OHP). The variability in the CPK response may have been due to the uneven numbers of males and females in this study, but the pattern of the responses was also similar to what has been reported previously. The greatest CPK response was 96 hours (four days) to 168 hours (seven days) post-exercise, which was later than the greatest perception of DOMS. The variability in the OH? response in this study may have been related to diet and the criteria for urine collection.  Other investigators have reported a lack of strong relationships among the responses of fatiguing eccentric exercise, a conclusion which supports the suggestion that more than one mechanism contributes to the characteristics of muscle injury after eccentric exercise. Because there was only one strong correlation among the outcome measures over the two exercise durations and over time {CPK and unpleasantness 168 hours (7 days) after the shorter exercise}, the results would suggest that, other than CPK and DOMS, the outcome measures examined in this study are not related.  35  References  1.  Armstrong RB. Mechanisms of exercise-induced delayed onset muscular  soreness: a brief review. Medicine and Science in Sports and Exercise 1984;16(6):529538.  2.  Gracely RH, Kwilosz DM. The descriptor differential scale: applying  psychophysical principles to clinical pain assessment. Pain 198835:279-288.  3.  Melzack R, Finch L. Objective pain measurement: a case for increased usage.  Physiotherapy Canada 1981 34(6)(Nov!Dec).  4.  Whitaker OC, Warfield CA. The measurement of pain. Hospital Practice  1988;(Feb): 15.  5.  Melzack R. Pain Measurement and Assessment. New York: Raven Press, 1983.  6.  Abraham WM. Factors in delayed muscle soreness. 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Effects of intensity and duration of muscular exercise on  delayed soreness and serum enzyme activities. Medicine and Science in Sports and Exercise 1983 ;15(6):461-465.  19.  Newham DJ, Jones DA, Ghosh G, Aurora P. Muscle fatigue and pain after  eccentric contractions at long and short length. Clinical Science 1988;74:553-557.  20.  Evans WI Exercise-induced skeletal muscle damage. The Physician and  Sportsmedicine 1987; 15( 1)(Jan): 88-100.  21.  Farrell M, Richards J. Analysis of the reliability and validity of the kinetic  communicator exercise device. Medicine and Science in Sports and Exercise 1986;18:4449.  22.  Reitz C, Rowinski M, Davies G. Comparison of Cybex II and KinCom reliability  on measures of peak torque, work and power at three speeds. Physical Therapy 1988;68(Abstract):782.  23.  Snow CJ, Johnson K. Reliability of two velocity-controlled tests for the  measurement of peak torque of the knee flexors and extensors during resisted muscle shortening and resisted muscle lengthening. Physical Therapy 1988; 68(Abstract):781.  38  24.  Hughes H, Hagen L, Sutton RAL. Liquid chromatographic determination of 4-  hydroxyproline in urine. Clinical Chemistry 1986;32(6): 1002-1004.  25.  Glass GV, Hopkins KD. Statistical Methods in Education and Psychology. (2 ed.)  Boston: Allyn and Bacon, 1984  26.  Appell HJ, Soares JM, Duarte JAR. Exercise, muscle damage and fatigue. Sports  Medicine 1992;13(2): 108-115.  27.  Newham DJ, Jones DA, Clarkson PM. Repeated high force eccentric exercise:  Effects on muscle pain and damage. Journal of Applied Physiology 1987;63: 1381-1386.  28.  Rodenburg JB, Bar PR, de Boer RW. Relations between muscle soreness and  biochemical and functional outcomes of eccentric exercise. Journal of Applied Physiology 1993 ;74(6):2976-2983.  29.  Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during  contractions: conditions of occurrence and prevention. Physical Therapy 1993;73(12):91 1-921.  30.  Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes after  strenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine 1992;119(2): 183  31.  -  188.  Clarkson PM, Byrnes WC, Gillisson E, Harper E. Adaptation to exercise-induced  muscle damage. Clinical Science 1987;73:383-386.  39  32.  Hortobágyi T, Denahan T. Variability in creatine kinase: methodological,  exercise, and clinically related factors. International Journal of Sports Medicine 1989;10(2):69 80. -  33.  Murguia MJ, Vailas A, Mandelbaum B, et al. Elevated plasma hydroxyproline A  possible risk factor associated with connective tissue injuries during overuse. American Journal of Sports Medicine 1988;16(6):660-664.  34.  Stauber WT. Personal communication June, 1993.  35.  Seaman R, lanuzzo CD. Benefits of short-term muscular training in reducing the  effects of muscular over-exertion. European Journal of Applied Physiology 1988;58(3):257-261.  36.  Horswill CA, Layman DK, Boileau RA, Williams BT, Massey BH. Excretion of  3-methylhistidine and hydroxyproline following acute weight-training exercise. International Journal of Sports Medicine 1988;9(4):245-248.  37.  Elam RP, Hardin DH, Sutton RAL, Hagen L. Effects of arginine and ornithine on  strength, lean body mass and urinary hydroxyproline in adult males. The Journal of Sports Medicine and Physical Fitness 1989;29(1):52-56.  40  CHAPTER TWO THE EFFECTS OF ECCENTRIC EXERCISE ON DELAYED MUSCLE SORENESS, MUSCLE STRENGTH AND FATIGUE, JOINT RANGE OF MOTION, AND  THE PRESENCE OF LEUKOCYTES IN EXERCISED MUSCLE  Introduction A recent review by Smith (1) has revisited the hypothesis that the sensation of muscle soreness is associated with the acute inflammatory response. This hypothesis was initially proposed in the 1970’s but at that time was not supported by research findings. Smith reviewed these earlier reports and suggested that some discrepancies were apparent and thus some of the conclusions were not well-founded. Similarities between DOMS and acute inflammation include pain, swelling, and the loss of function as measured by muscle strength. Additionally, evidence of cellular infiltrates, such as macrophages, has been reported, which have been noted to be present at the site of injury in animals from one to three days (2, 3). Other indicators of inflammation such as the presence of fibroblasts (2) and increased lysosomal activity (4, 5) have also been found. Progression of the size of the lesion up to 48 hours has been observed (6), with signs of healing at 72 hours (2, 7).  In a study of patients with inflammatory muscle disease, Yonker and colleagues (8) suggested that technetium-99m pyrophosphate (Tc-PYP) muscle scans are useful in establishing sites of inflammation. Their results indicated that five of six patients with active inflammatory muscle disease had positive scans, while nine patients without active disease had negative scans. They concluded that the muscle scans separated the acute inflammatory disease from inactive disease more consistently than muscle tenderness, increased CPK, muscle weakness or EMG.  41  Jones and colleagues (9) and Newham and colleagues (10) have reported on the utilization of Tc-PYP injections and scans after two different eccentric exercise regimes. They found an increased uptake of Tc-PYP into the exercised muscles and suggested that there may be a correlation with increased CPK but they did not assess the correlation statistically. Oiily two or three subjects participated in each of the protocols. They also reported that little is known about the mechanism by which Tc-PYP is taken up into muscle, although some have proposed an association between calcium accumulation and Tc-PYP concentration (11). Thus, Tc-PYP may not be the best choice of an investigative technique to assess the underlying mechanism of muscle soreness, especially if inflammatory mediators are associated with the soreness.  Technetium-99m (Tc-99m) is a radioisotope that has certain advantages for clinical use it has a short half-life of six hours, it has very good imaging characteristics, and there -  is no beta radiation resulting in a lower radiation dose (12). Within the last two decades it has also been used clinically as a radio label for white blood cells (WBC) to localize infections or abscess formation. As Evans and Cannon (13) and Smith and colleagues (14) have suggested that an increase in leukocytes in the first 24 hours after eccentric exercise would be indicative of an acute phase inflammatory response, it follows that by labelling WBC, this may provide evidence of acute inflammation in muscle if there is an increased number of leukocytes within the muscle after eccentric exercise.  Some investigators (4, 15) have reported upon decreased range of motion (ROM) after eccentric exercise and have suggested that this may be due to muscle shortening as a  result of calcium accumulation in damaged fibres.  However, others (16, 17) have  concluded that it might be edema within the perimuscular connective tissue that is responsible for the restricted movement. Two studies (16, 18) have provided evidence that the pressure from the increased fluid may also cause sensitization of the pain receptors  42  resulting in tenderness and muscle soreness. If edema is responsible for decreased ROM and sensitization of the pain receptors, then perception of DOMS and ROM should be correlated.  Faulkner and colleagues (19) have suggested that the initial loss of force, immediately after the fatiguing eccentric exercise, is a function of both fatigue and injury to the muscle tissue. They believe that recovery from fatigue occurs over the next three hours and is complete by 24 hours. Other investigators have studied the force/frequency characteristics of eccentrically exercised muscles (20, 21). It has been found that there is a decrease in force generation at low frequencies of stimulation and that this low frequency fatigue, or what may also be termed long-lasting fatigue, is more common with eccentric exercise than concentric.  It has also been noted that this long-lasting fatigue is most common  immediately after eccentric exercise (21). Jones and colleagues (22) have suggested that mechanical damage to the muscle caused by the high forces of eccentric activity causes the long-lasting fatigue.  Power spectrum analysis has been widely employed to analyze muscle fatigue (2325). It has been suggested that one of the factors that contributes to muscle fatigue is a  slowing of the propagation velocity of the muscle fibre action potentials (24).  This  decrease in the propagation velocity can contribute to a change of shape of the surface electromyograph (EMG) signal during a prolonged isometric contraction (26). The change in EMG signal shape is measured as a shift in the frequency of the signal to a lower frequency (24). The power spectrum describes the relationship between signal amplitude and signal frequency (24). The median frequency, which is a quantitative value, is an index of the frequency shift (25) and thus, a measure of the slowing of the action potential propagation velocity.  43  In order to determine whether or not leukocytes were present in muscle following eccentric exercise, a nuclear medicine technique was used to label WBC, and to monitor their location over 24 hours. To determine whether or not other symptoms of acute inflammation were present in the muscle, the perception of muscle soreness and unpleasantness was assessed, ROM of the knee joint was measured, and muscle strength was determined over 72 hours. In addition, to determine whether muscle fatigue was present after the exercise stimulus, assessment of muscle fatigue, utilizing power spectrum analysis, was conducted over 72 hours following the eccentric exercise.  Purpose It was the purpose of this study to examine whether there was increased presence of Tc-99m WBC in the exercised muscle, compared to the contra-lateral non-exercised muscle over time. In addition, it was the purpose of this study to determine the time course and relationships over time among DOMS, eccentric torque of the quadriceps muscles, muscle fatigue of the quadriceps, range of motion (ROM) of the knee, and the presence of Tc-99m WBC in the exercised muscle.  Research Hypotheses  1. There will be greater presence of Tc-99m WBC in the exercised muscle compared to the contra-lateral non-exercised muscle. 2. The greatest perception of DOMS (measured as intensity of soreness and unpleasantness) will occur between 24 and 48 hours following the exercise stimulus, at the same time as the greatest presence of Tc-99m WBC and the greatest loss of ROM. 3. The greatest decline in eccentric torque will occur in the first 24 hours following the exercise stimulus, at the same time as the onset of muscle fatigue, but before the  44  greatest perception of DOMS. Muscle fatigue will be measured as the median frequency of the power spectrum.  Methods and procedures Subjects  Twelve subjects volunteered for this study 11 females and one male. The data from -  one female subject was not included because one nuclear medicine scan time was missed and other test times became different from the protocol of this study. It was decided to analyze only the data from the female subjects so that the subject group was more homogeneous. Therefore, ten female subjects between the ages of 20 and 33 years participated in this study. Individuals were excluded from the study if they were pregnant, missed their last menstruation or were breast-feeding, or if they were engaged in recreational exercise of more than six hours/week or involved in competitive sport, or if they had cardiovascular, neurological or musculoskeletal conditions that may have compromised their ability to perform the testing procedures or which may have posed a hazard to the individual as identified by their physician (i.e..  -  cardiac disease, a chronic  neurological disease, degenerative knee joint disease, a recent soft tissue injury of the lower extremity). Approval for this study was granted by the Clinical Screening Committee for Research Involving Human Subjects at the University of British Columbia.  Protocol  Following informed consent, subjects completed baseline measures of the Descriptor Differential Scale (DDS) describing DOMS, eccentric torque of the right quadriceps, a power spectrum analysis of the right quadriceps, and range of motion (ROM) of the right knee. Fifty milliliters (ml) of blood were taken by vempuncture. The WBC were sepamted  45  and labelled with Tc-99m HMPAO {(RR,SS)-4,8 diaza-3,6,6,9-tetramethyl undecane 2,10-dione bisoxime} (See Appendix Four). The labelled WBC were re-introduced intravenously to the subjects immediately prior to the exercise stimulus. Following the exercise stimulus (300 repetitions), the DDS questionnaire, eccentric torque, power spectrum analysis and ROM were repeated at two hours, four hours, 20 hours, 24 hours, 48 hours and 72 hours. An extra test time for eccentric torque occurred at 0 hour at the end of the exercise stimulus. Bilateral scans of the quadriceps muscles (anterior and lateral) were taken at two hours, four hours, 20 hours and 24 hours after the exercise stimulus.  Due to the decay of Tc-99m over 24 hours the data collection for Tc-99m WBC was limited to 24 hours, while data collection for the other dependent variables continued for 72 hours. As well, there was no pre-test for the presence of Tc-99m WBC but rather the exercise leg was compared to the non-exercise leg. This reduced the number of scan times.  Methods  Descriptor Differential Scale  The DDS was measured as described in Chapter One.  Eccentric/Concentric Torque of the Quadriceps Muscles  Eccentric torque was measured the same as in Chapter One, except that data were also collected during the last set of the exercise stimulus (0 hour). In this case, repetitions two to four were analyzed as the average eccentric torque of the knee extensors.  46  Power Spectrum Analysis  The subjects were seated on the KinCom as described for the Eccentric/Concentric Torque of the Quadriceps Muscles. The knee was positioned at 90 degrees of knee flexion, the center of rotation of the KinCom was positioned opposite the knee joint line and the lever arm was adjusted so that the resistance pad was placed on the distal aspect of the tibia as stated previously.  The skin was cleaned with an alcohol swab and surface EMG electrodes (Medi Trace silver/silver chloride, circular, 1 cm radius) were placed over the motor points of the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM) muscles according to Delagi and colleagues (27). The interelectrode spacing was 2.5 cm. A ground electrode was placed over the wrist. Electrodes were placed on the skin with electromedical gel between the skin and the electrodes. Subjects maintained a maximum isometric contraction for 60 seconds. A ten-second submaximal isometric contraction preceded the one minute fatiguing contraction as a warm-up.  The amplifier (custom-made) gain was set at 5000. The input impedance was 10 megohms and the common mode rejection ratio (CMRR) greater than 100 dB. The raw signal was amplified and then passed through a band pass filter with a frequency range of 28-500 Hz. The force and EMG signals were simultaneously collected by the computer for spectral analysis. This analysis entailed partitioning the EMG data into overlapping foursecond segments with each successive segment starting two seconds later than the previous segment. Spectral estimates of each segment were then calculated. The median frequency served as the measure by which muscle fatigue was calculated. The median frequency of each four-second segment was plotted against time and a linear regression line was fitted to the 28 data points. The slope of the plot was considered a quantitative measure of muscle  47  fatigue. The steeper the negative slope of the plot, the greater the muscle fatigue because as muscle fatigues over time the spectral frequency shifts to a lower frequency.  (See  Appendix Three for further description of the power spectrum analysis).  Range of Motion  Each subject was positioned supine on a plinth. The centre of the goniometer was positioned opposite the lateral aspect of the right knee joint line. Active range of motion (ROM) of the knee was measured with a plastic goniometer from full active extension to full active flexion with the hip also fully flexed. ROM represented the painfree ROM that the subject could actively attain. The measured ROM did not differentiate between lack of full extension and/or loss of full flexion. A previous study by Rothstein and colleagues (28) reported the intra-tester reliability of goniometric measures of knee flexion and extension of 12 patients to range between 0.91 and 0.99 with a plastic goniometer. In their study using the means of multiple measurements only improved the correlations slightly.  The Exercise Stimulus  The exercise stimulus was the same as outlined in Chapter One, except that in this study the exercise stimulus required completion of 300 maximal eccentric contractions only. The subjects completed 30 sets of 10 repetitions with each set beginning every minute for 30 minutes. This allowed a 20 second rest between each set.  Radio-isotope Investigation of Acute Inflammation  The WBC were separated from the blood sample and labelled with Tc-99m HMPAO according to procedures in the Nuclear Medicine Department, Vancouver Hospital  48  (Appendix Four). Immediately before the exercise stimulus the labelled WBC were re injected intravenously into the subjects. Following the exercise stimulus, bilateral scans of the quadriceps muscles (anterior and lateral views) were taken at two, four, 20 and 24 hours. As the half-life of Tc-99m is six hours and more than 90% of it has decayed by 24 hours (12), the scan times were limited to within 24 hours. After the series of four scans was completed, computer analysis of regions of interest (ROl) was used to determine the count/pixel of gamma radiation. Four areas of the quadriceps muscle were chosen as the ROT  -  the antero-distal aspect from the lateral view, the anterior aspect from the lateral  view, the medial aspect from the anterior view and the lateral aspect from the anterior view (Figure 4).  The analyses of the ROT were undertaken on three separate occasions by two scorers (observers). The ROT were carefully drawn to avoid the femur and the femoral circulation. The mean count/pixel of the three analyses were then calculated as well as the standard deviation (SD). The coefficient of variation (CV) was calculated as the measure of interobserver variability (Appendix Four). Next, the background, or noise, of the ROT of the exercise leg was subtracted. Tt is common in clinical practice to use the contra-lateral side of an organ or segment of the body to represent the background, and in this case the respective ROT of the non-exercise leg was subtracted from the ROT of the exercise leg. Tt is also necessary to correct for the physical decay of the Tc-99m and this was done using a decay table to correct the count/pixel at each scan time (Appendix Four). Finally, as dosages of Tc-99m HMPAO varied between subjects, the counts/pixel were normalized to the peak response of each subject and presented as a percentage so that responses could be compared between subjects who had different baselines (i.e.. different dosages of Tc-99m -  HMPAO).  49  Figure 4. Regions of Interest for Radio-Isotope Scanning. The anterior view of both thighs is shown on the left. In the middle is the lateral view of the right thigh (exercise leg). On the right is the lateral view of the left thigh (non-exercise leg). The four regions of interest are identified on the figure.  (3 cD  51  Statistical analysis  A one-way repeated measures multivariate analysis of variance (MANOVA) was used on each of the dependent variables (DDS intensity of soreness score, DDS unpleasantness score, eccentric torque, slope of the median spectral frequency of VL and VM, and ROM) to test for significant differences over time. A one-way repeated measures MANOVA was used to examine the differences in the presence of Tc-99m labelled WBC in the quadriceps muscles over time and between legs. As the “constant”, which is one source of variation in the MANOVA analysis, represents the average of the means over the four test times and tests whether or not the average of the means is different from zero, and in our data preparation each ROl of the non-exercise leg was subtracted from the ROT of the exercise leg, it was appropriate to use the “constant” to determine whether or not the ROT of the exercise leg was significantly different from zero and thus, significantly different from the non-exercise leg. As in the analyses of the other dependent variables, the source of variation “time” was used to determine the significant differences among the four time periods over time.  Post hoc analyses examined contrasts between test times for all dependent variables and at each test time compared to the pre-test for DOMS, eccentric torque, muscle fatigue and ROM. Only those variables that were significant over time were examined as the multivariate F test is more robust than the univariate post hoc tests (29). Correlation analyses (Pearson product moment correlation) were used to determine relationships among DOMS, eccentric torque, muscle fatigue and ROM. The significance level was set at p<O.O5.  52  Results There was a significantly greater presence of Tc-99m WBC in the four ROl of the exercised muscle compared to the contra-lateral non-exercised muscle (p<0.0O1) (Table 8). The greatest presence at two hours post-exercise was in the antero-distal ROT and the presence remained at approximately the same level over 24 hours (Figure 5A), while the presence of Tc-99m WBC gradually increased over 20 and 24 hours in the other ROT (Figure 5).  All of the dependent variables changed significantly over time (p<0.05) except the slope of the median spectral frequency for VM and the presence of Tc-99m WBC in the antero-distal ROl (Tables 7, 8). Intensity of soreness and unpleasantness were highest at 24 hours post-exercise (Figure 7). The greatest loss of ROM also occurred at 24 hours post-exercise (Figure 8). Eccentric torque was lowest at 0 hour, recovered, and then declined again at 20 to 24 hours post-exercise (Figure 6). The slope of the median frequency of vastus lateralis declined at two hours post-exercise (Figure 9). The greatest presence of Tc-99m WBC occurred at 20 hours post-exercise in the antero-distal ROT and the lateral ROT (Figure 5A and D). The greatest presence of Tc-99m WBC in the anterior ROI and the medial ROT was at 24 hours post-exercise (Figure SB and C). The means and standard deviations for each of the dependent variables over time are presented in Tables 5 and 6.  The contrasts between the pre-test and each subsequent testing time were all significantly different (p.<0.05) for intensity of soreness, unpleasantness and ROM (Table 9, Figures 7, 8). Each testing time up to and including 24 hours was significantly different from the pre-test for eccentric torque (Table 9, Figure 6). Between testing times, there were four significant contrasts between time periods for eccentric torque indicating the  106.90 ±29.26 139.50 ± 4.97  -5.87 ± 5.6 -8.77 ± 2.24  -0.21 tO.13 -0.20 ± 0.25  73.90±29.11 133.00 ± 5.87  +  3.60  -0.28±0.13 -0.23 ± 0.20  89.4±28.12 137.0 ± 5.87  -2.21  6.88 ± 6.50  4 Hours  133.80 ± 6.32 -0.20± 0.10 -0.18 ± 0.14  93.70 ±39.78  10.26 ± 4.80 0.69 ± 6.26  48 Hours  -0.23 ±0.12  -0.29 ± 0.14  86.00±32.00 136.80 ±6.16  -3.63 ±3.34  6.44 ± 6.90  2 Hours  11.11 ± 3.54 2.11 ± 4.08  24 Hours  65.90 ±22.12  0 Hour  137.80 ± 5.01 -0.23 ±0.11 -0.24 ±0.19  94.20 ±35.27  4.94 ± 8.42 -2.60 ± 5.47  72 Hours  -0.22 ± 0.12 -0.22 ± 0.20  1.80 ± 3.66 75.70 ±28.74 134.50 ± 5.50  8.94 ± 5.73  20 Hours  Median Frequency  Data are means ± SD for ten female subjects. *=Range of Motion,+=Vastus Lateralis Median Frequency and Vastus Medialis  VLMedFreq(slope) VM Med Freq (slope)  ROM (degrees)  Torque (N.m)  Unpleasantness  Intensity of Soreness  VL Med Freq (slope) -0.25 ± 0.12 VM Med Freq (slope) -0.22 ± 0.11  Torque(N.m) ROM* (degrees)  Unpleasantness  Intensity of Soreness  Pre-test  INDIRECT MEASURES OF EXERCISE-INDUCED MUSCLE INJURY BEFORE AND AFTER ECCENTRIC EXERCISE  TABLE5  01 (.3  32.77 ±22.54 45.53 ±42.19  Medial  41.01 ±32.14 40.68 ±44.81  68.54 ± 21.03 42.80 ±28.31  4 Hours  65.49 ±34.07 90.59 ±16.50  88.19 ± 25.67 68.71 ±33.07  20 Hours  Data are means ± SD for ten female subjects. The regions of interest were measured as counts/pixel.  Lateral  Anterior  77.55 ± 21.22 52.01 ±32.05  2 Hours  Antero-distal  Regions of Interest  82.76 ±15.70  77.24 ±28.18 81.42 ±21.14 90.76 ± 16.77  24 Hours  THE PRESENCE OF WHITE BLOOD CELLS IN FOUR REGIONS OF INTEREST IN THE EXERCISED MUSCLE AFTER ECCENTRIC EXERCISE  TABLE6  c-fl  55  TABLE7 MANOVA TABLES FOR THE INDIRECT MEASURES  Source (of Variation)  df  MS  F  Intensity of Soreness Within Subjects Time  54  23.67  6  326.55  54  12.55  6  145.34  63  225.54  7  1758.21  54  8.42  6  55.33  54  0.00  6  0.01  54  0.01  6  0.00  13.79*  Unpleasantness Within Subjects Time  11.58*  Eccentric Torque Within Subjects Time  7.80*  Range of Motion Within Subjects Time  6.57*  Vastus Lateralis Median Frequency Within Subjects Time  5.31*  Vastus Medialis Median Frequency Within Subjects Time  *  001 . 0 p<  0.37  56  TABLE8 MANOVA TABLES FOR THE PRESENCE OF WHITE BLOOD CELLS IN FOUR REGIONS OF INTEREST  df  Source (of Variation)  MS  F  Antero-Distal Region of Interest 9  578.18  1  242625.79  27  589.25  3  646.57  9  763.55  1  149993.91  27  868.41  3  2960.73  9  885.95  1  132274.15  27  698.71  3  6844.49  Within Subjects  9  2075.06  Constant  1  168432.38  27  744.10  3  6470.23  Within Subjects Constant Within Subjects Time  419.64* 1.10  Anterior Region of Interest Within Subjects Constant Within Subjects Time  196.44* 3.41k  Medial Region of Interest Within Subjects Constant Within Subjects Time  149.30* 9.80*  Lateral Region of Interest  Within Subjects Time *  +  p< 0 . 0 01 p<0.05  81. 17* 8.70*  57  TABLE9 POST HOC CONTRASTS OF THE INDIRECT MEASURES Umvariate F  p  pre-2hr  17.96  0.002*  pre-4hr  20.85  0.001*  pre-2Ohr  27.12  0.001*  pre 24 hr  43.54  pre-4Shr  37.64  <.001 * <.001*  pre-72hr  15.89  0.003*  2hr -4 hr  0.25  0.631  4hr-2Ohr  4.92  0.054  2Ohr-24hr  2.21  0.171  24hr-48hr  0.62  0.450  48 hr -72 hr  4.76  0.057  pre-2hr  19.94  0.002*  pre-4hr  27.70  0.001*  pre-2Ohr  69.48  <.001*  pre-24hr  44.22  <.001*  pre-48hr  16.50  0.003*  pre-72hr  12.16  0.007*  2hr-4hr  16.45  0.003*  4hr-2Ohr  9.95  0.012*  2Ohr-24hr  0.76  0.789  24hr-48hr  1.64  4Bhr-72hr  1.96  0.233 0.195  Source Intensity of Soreness df 1,9  -  Unpleasantness  Eccentric Torque  df 1,9  df 1,9  pre 0 hr  45.49  pre-2hr  6.70  -  <.001 * 0.029*  58  Umvariate F  Source  p  Eccentric Torque cont. pre-4hr  5.54  0.043*  pre-2Ohr  17.52  0.002*  pre 24 hr  23.67  <.001 *  pre -48 hr  1.72  0.222  pre-72hr  2.23  0.169  Ohr-2hr  6.30  0.033*  2hr-4hr  0.81  4hr-2Ohr  12.83  0.392 0.006*  2Ohr-24hr  0.18  24hr-48hr  16.11  0.685 0.003*  48 hr -72 hr  0.02  0.884  pre-2hr  5.20  0.048*  pre-4hr  9.00  0.015*  pre-2Ohr  15.00  0.004*  pre-24hr  37.10  <.001*  pre-48hr  16.80  0.003*  pre-72hr  5.19  0.049*  2hr-4hr  0.07  0.801  4 hr -20 hr  3.46  0.096  2Ohr-24hr  1.98  0.193  24 hr 48 hr  0.32  48hr-72hr  16.00  0.583 0.003*  -  Range of Motion  df 1,9  -  Vastus Lateralis Median Frequency  df 1,9  pre-2hr  3.17  0.109  pre -4 hr  1.45  0.260  pre-2Ohr  1.15  0.311  pre-24hr  1.76  0.217  59  Umvariate F  Source  p  Vastus Lateralis Median Frequency cont.  *  pre-48hr  2.32  0.162  pre-72hr  0.64  0.444  2hr-4hr  0.54  4hr-20hr  13.46  0.482 0.005*  2Ohr-24hr  1.22  0.299  24hr-48hr  .0.01  0.937  48 hr-72 hr  1.86  0.206  significant differences between tests  60  Figure 5. The Presence of White Blood Cells in the Four Regions of Interest in the Exercised Muscle. Values are means ± SD. The presence of white blood cells in the exercised muscle was significantly greater than in the contra-lateral nonexercised muscle (p<O.OO1) in all four regions. A. Antero-Distal Region of Interest. B. Anterior Region of Interest. Significant differences over time p<O.O5. C. Medial Region of Interest. Significant differences over time p<O.OO1. D. Lateral Region of Interest. Significant differences over time p<O.OO1. *  Significantly different this test to previous test p<O.O1  I  be  a  0  z  -J  Ui 1—i  0 C-)  Cs)  B  A  80  100  0  20  40  60  80  100  0  0  4.  4.  S  I  5  I I  10 15 TIME (Hours)  I  10 15 TIME (Hours)  I  20  20  I  F  S  25  25  Figure 5  a  0  Ui tJ  Li 0  a  U) I  be  D  C  0  20  40  60  80  100  80  100  0  0  5  5  10 15 TIME (Hours)  10 15 TIME (Hours)  20  20  25  25  62  Figure 6. Characteristics of Eccentric Exercise: Eccentric Torque. Values are means L SD. There were significant differences over time p<O.OO1. *  Significantly different from the pre-test p<O.05.  the pre-test p<O.O 1. ++  **  +  Significantly different from  Significantly different from the pre-test p<O.OO1.  Significantly different this test to previous test p<O.05.  different this test to previous test p<O.O1.  Significantly  (J  C  0  m  -1  r’J  —1  r’J  0  0  (D  1  0 0  r’J  0  -  0  0  ZD  0 0  -  ECCENTRIC TORQUE (Nm)  -  0  r’)  0  .  -  (D  O•) U)  64  Figure 7. Characteristics of Eccentric Exercise: Muscle Soreness. Values are means ± SD. There were significant differences over time p<O.OO1. *  Significantly different from the pre-test p.cO.O 1.  +  Significantly different from  the pre-test p<O.OO . 1 A. Intensity of Soreness B. Unpleasantness. ++  **  Significantly different this test to previous test p<O. 05.  Significantly different this test to previous test p.<O.O1.  U,  1  0 C  N.)  o  N.)  N.)  -o  —  -  *1  -.  —  —  •  -  -  -  -  -  -  --  ...  r  I’  *  S  I  S  I —  +  ————r—-—-r  I  I  I  I  I  5  •  I  S  I  K  a  a  a  I  a a  +  ji..—..r  o  .  .  I  MEAN SCORE  .  .  F—  I  j  I  ..J  I  i__  U,  -,  0 C  m  co  —-  C)  -‘  -o  -  —  ..  -,  -  --  .  -  0  I  7  I  ...  .  --  .  I  o  I  ——  *1  I  +  .  .  I  MEAN SCORE  I  1  +  1  I  I  I  a  I  ....  .  o  I  .L_  —  —  —  > (1)  0,  0  66  Figure 8. Characteristics of Eccentric Exercise: Range of Motion of the Knee. Values are means ± SD. There were significant differences over time p<O.OO1. *  Significantly different from the pre-test p<O.05.  the pre-test p<O.O 1. ++  **  +  Significantly different from  Significantly different from the pre-test p<O.OO1.  Significantly different this test to previous test p<O.O1.  -‘  0  m  N)  N)  0  NJ  Ni  -  --  --  •--  --  rJ LI,  .  + -  -  __.._.____._._l  -  0  Lu  -  +  I  1  I  L&) U,  -  --  -  I  I  I  0  I  .  ____[  U,  -  RANGE OF MOTION (degrees)  (  OD  ‘-I  68  Figure 9. Slopes of the Median Frequencies of Two of the Quadriceps Muscles. Values are means ± SD. A. Median Frequency of the Vastus Lateralis Muscle. Significant differences over time p<O.OO1.  *  Significantly different this test to the previous test p<O.Ol.  B. Median Frequency of the Vastus Medialis Muscle.  p  (11  I  Cn  Cx) ci,  p  I;  U,  -  I  P  I::  I  U,  0 U,  i  -  CO  U)  I  0  0  *  T  U,  I\)  P  II  U,  0  I  SLOPE OF THE MEDIAN FREQUENCY  I  SLOPE OF THE MEDIAN FREQUENCY  Ci,  0  I  P CD  C  70  changes that were occurring in muscle strength over time (Table 9, Figure 6). The significant contrasts for unpleasantness were between two and four hours and between four and 20 hours post-exercise, as the perception of unpleasantness was increasing (Table 9, Figure 7B). For ROM, there was significant change between 48 and 72 hours postexercise as ROM was increasing again (Table 9, Figure 8). The only significant contrast for VL median frequency was between four and 20 hours as the slope of the median frequency was increasing (Table 9, Figure 9A). There was only one significant contrast for the lateral ROl between four and 20 hours post-exercise as the presence of Tc-99m WBC was increasing (Table 10, Figure 5D).  Table 11 is a summary of the correlations among the dependent variables, presented as correlation matrices. Although it was originally proposed to correlate the other dependent variables with the presence of Tc-99m WBC, these correlations have not been reported because the Tc-99m WBC data was normalized. Upon plotting some of the data it was noted that the normalized values of 100% created a “ceiling effect” which did not represent the range of values for each subject. However, the non-normalized data did not account for the differences between the dosages of Tc-99m HMPAO.  ROM and unpleasantness were significantly correlated at 20 hours post-exercise (r=-0.77). Other significant correlations between eccentric torque and vastus lateralis median frequency (r= -0.65) and vastus medialis median frequency (r= -0.68) were found at two hours post-exercise, and again at 20 hours for vastus medialis median frequency (r= -0.64).  Significant correlations were found between intensity of soreness and  unpleasantness at the pre-test (r=0.73) and at 72 hours (r=0.85) following the exercise stimulus (Table 11).  71  TABLE 10 THE PRESENCE OF WHITE BLOOD CELLS IN THE FOUR REGIONS OF INTEREST: POST HOC CONTRASTS Umvariate F  Source Anterior Region of Interest  df 1,9  2hr-4hr  1.09  0.323  4 hr -20 hr  4.22  0.070  2Ohr-24hr  1.11  0.320  2hr-4hr  1.07  0.328  4hr-2Ohr  3.58  0.091  2Ohr-24hr  2.97  0.119  2hr-4hr  0.24  4hr-2Ohr  14.74  0.637 0.004*  20 hr 24 hr  0.77  0.402  Medial Region of Interest  Lateral Region of Interest  *  p  df 1,9  df 1,9  significant differences between tests  72  TABLE 11 RELATIONSHIPS AMONG MUSCLE SORENESS, MUSCLE STRENGTH AND FATIGUE, AND RANGE OF MOTION AT EACH TEST TIME CORRELATION MATRICES Test Times (hours) Pre-test  2  4  20  24  48  72  Intensity of Soreness Test Times (hours) Unpleasantness 0.73* Pre-test 2 4 20 24 48 72 Eccentric Torque Pre-test 0.54 2 4 20 24 48 72 Pre-test 2 4 20 24 48 72 Pre-test 2 4 20 24 48 72  0.52  0.50 0.18 -0.18 0.59 0.85*  0.51 0.25 0.01 -0.41 -0.53  0.04  Median Frequency of Vastus Lateralis -0.35 -0.44 O.71* -0.44 -0.36 -0.23 -0.56 Median Frequency of Vastus Medialis 0.11 -0.17 -0.46 -0.24 -0.28 -0.35 -0.47  73  Test Times (hours) Pre-test  2  4  20  24  48  72  Intensity of Soreness cont. Test Times (hours) Range of Motion Pre-test -0.59 2 4 20 24 ‘48 72  0.04 0.11 -0.21 0.38 -0.33 0.02 Unpleasantness  Eccentric Torque Pre-test 0.55 2 4 20 24 48 72 Pre-test 2 4 20 24 48 72 Pre-test 2 4 20 24 48 72 Pre-test 2 4 20 24 48 72  0.19 0.13 0.22 0.38 -0.30 -0.20  Median Frequency of Vastus Lateralis -0.11 -0.27 -0.45 -0.24 0.07 0.02 -0.48  Median Frequency of Vastus Medialis 0.16 0.07 0.04 -0.25 0.30 -0.03 -0.30 Range of Motion -0.55 -0.41 -0.09 O.77* -0.30 -0.47 -0.28  74  Test Times (hours) Pre-test  2  4  20  24  48  Eccentric Torque Test Times (hours) Median Frequency of Vastus Lateralis Pre-test -0.38 .0.65* 2 4 -0.21 20 -0.48 24 -0.001 48 -0.25 72 Median Frequency of Vastus Medialis Pre-test -0.05 .0.68* 2 4 -0.22 .0.64* 20 24 -0.32 48 -0.03 72 Pre-test 2 4 20 24 48 72  *  -0.26  -0.31  Range of Motion -0.29 -0.07 -0.08 -0.04 0.02 -0.01 0.002  Range of Motion Median Frequency of Vastus Lateralis Pre-test 0.24 2 0.07 4 0.07 20 0.24 24 -0.06 48 0.14 72 Pre-test 2 4 20 24 48 72  72  -0.02  Median Frequency of Vastus Medialis -0.22 0.36 0.15  p< 0 . 0 5  0.11 0.18 -0.23 0.03  75  Discussion  Together with indirect measures of muscle damage, a technique to measure the muscle damage directly should also be employed (19). In this study the presence of Tc 99m WBC in the exercised muscle compared to the contra-lateral non-exercised muscle was monitored over 24 hours following eccentric exercise. In all of the ROl the presence of Tc 99m WBC in the exercised quadriceps muscles was significantly greater (p<0.0O1) than in the contra-lateral non-exercised muscle (Table 8). The presence of Tc-99m WBC also increased up to 20 and 24 hours post-exercise, except for the antero-distal aspect which remained high from two to 24 hours post-exercise (Figure 5). In addition, there was a significant increase in the presence of Tc-99m WBC between two hours and 20 hours postexercise for the lateral aspect.  However, because the presence of Tc-99m WBC was not  monitored beyond 24 hours, it would be premature to conclude that the greatest presence of WBC in the muscle occurred at 20 and 24 hours following eccentric exercise.  The significance of these findings are two-fold.  First, the results show that  inflammatory cells were present in the exercised muscle of humans in the 24 hours following eccentric exercise, similar to what occurs in animals (2). In this study the Tc 99m WBC were found to be in significantly greater numbers in the exercised quadriceps muscles compared to the contra-lateral non-exercised muscles over the first 24 hours post exercise.  Secondly, the greatest presence of Tc-99m labelled WBC immediately post-exercise was into the antero-distal ROl of the exercised muscle (Figure 5A). However, it was evident that the WBC were present throughout the muscle in increasing numbers over 24 hours (Figure 5). This nuclear medicine technique allowed a quantitative evaluation of the  76  widespread distribution of the exercise-induced muscle damage, as reflected by the presence of inflammatory cells in the exercised muscle, compared to the smaller sample that would have been evaluated from microscopic examination of human muscle biopsies. Although the WBC presence was greatest in the antero-distal ROl of the exercised muscle two hours post-exercise and this continued to be the most visually obvious site of increased Tc-99m WBC over 24 hours, the increased WBC in the other ROT also suggests evidence of inflammation throughout the entire quadriceps muscle.  Due to the normalized data, it was not possible to correlate the presence of Tc-99m WBC with the other dependent variables. However, during the data collection subjects were asked to subjectively report the location of their muscle soreness by indicating on a body diagram where they felt the soreness. At two hours post-exercise nine out of the ten subjects reported their soreness in a distal location on the right anterior thigh. By 24 48 -  hours post-exercise all of the subjects reported soreness proximally through the quadriceps muscles. These reports are similar to the timing of the increasing presence of Tc-99m WBC in the four ROT. Although this is anecdotal information, Newham and colleagues (21) have also reported tenderness beginning medially, laterally and distally and then becoming more diffuse throughout the quadriceps muscles by 24  -  48 hours after the  exercise. These findings suggest further study. If it is possible to keep the dosages of the radio-isotope constant, then it would be possible to compare the measure of acute inflammation to the other dependent variables, such as intensity of soreness and eccentric torque, following eccentric exercise.  As indicated in the protocol, additional eccentric torque data were collected at the end of the exercise stimulus (0 hour) as well as at the other time periods of the protocol. By collecting data between the end of the exercise stimulus and 24 hours after, it can be seen that there was some recovery of torque after the first decline at 0 hour but then it declined  77  again at 20 and 24 hours post-exercise (Figure 6). Faulkner and colleagues (19) have reported this pattern of response in mice but to date this has not been reported in humans. Most other investigators have reported the greatest decline in force in humans immediately following the exercise with recovery at 24 hours and onwards (4, 22, 30), but in these previous studies, data were not collected between one hour and 24 hours post-exercise.  The second decline in eccentric torque in this study occurred from 20  -  24 hours  (Figure 6). The peak intensity of soreness and unpleasantness occurred at the same time. This suggests that the intensity of soreness and unpleasantness may be related to a secondary response to the original injury to the muscle but there were no significant correlations between eccentric torque and either intensity of soreness or unpleasantness (Table 11). The different response patterns during the first 24 hours between eccentric torque and either intensity of soreness or unpleasantness may explain why no studies have found a definitive relationship between DOMS and muscle strength.  The subjects’ perception of intensity of soreness and unpleasantness both peaked at 24 hours following the exercise stimulus (Figure 7). Although every time period was significantly different from the pre-test, there was little difference between successive responses from two to 72 hours for intensity of soreness and from 20 to 72 hours for unpleasantness. Reports in the literature have stated that the greatest sensation of soreness occurs between 24 and 48 hours in the quadriceps muscles (21) and between 48 and 72 hours in the elbow flexors (30, 31). In this study three additional time periods of data were collected between the pre-test and the usual reporting time of 24 hours. The responses at two, four and 20 hours after the eccentric exercise confirmed that the intensity of soreness and unpleasantness progressively increase for up to 24 hours. Previous testing (Chapter One) revealed that both intensity of soreness and unpleasantness responses had returned to  78  baseline levels by seven days but because the protocol ended at 72 hours in this study neither had returned to the pre-test levels.  Another classic symptom of acute inflammation is the formation of edema or swelling (1). Based on the hypothesis that swelling within the muscle tissue will result in decreased ROM and increased discomfort within the first 48 hours of the eccentric exercise (16), the results of this study indicate significant loss of ROM up to 24 hours post-exercise (Figure 8) a pattern similar to the inverse response of the intensity and unpleasantness of soreness (Figure 7).  Although the changes in ROM were not significantly correlated with intensity of =O.59) between ROM and 2 soreness, there was a significant inverse correlation (r=-O.77, r unpleasantness at 20 hours (Table 11). The higher the unpleasantness score for a subject, the less the ROM at the knee. The single significant correlation should be interpreted cautiously but by assessing more than one dimension of the discomfort of muscle soreness, additional information regarding the onset of muscle soreness may be gathered.  As the pain receptors are within the connective tissue surrounding the muscle and group IV sensory fibres terminate as free nerve endings in this same region (5), the relationship between decreased ROM and unpleasantness lends support to the mechanical effect of swelling within the connective tissue causing increased pressure and thus discomfort. The lack of a relationship between eccentric torque and either intensity of soreness or unpleasantness (Table 11) provides some evidence that the pain receptors in the connective tissue appear to be reflecting the muscle injury differently than the functional response of the muscle itself.  Stauber (5) believes that the pain receptors may be  responding to swelling of the endomysium and perimysium. Faulkner and colleagues (19) suggest that the changes in muscle strength are reflecting the damage to the myofiber.  79  Finally, in order to determine whether or not the initial loss of strength immediately after the strenuous eccentric exercise was due to muscle fatigue, a power spectrum analysis of the superficial quadriceps muscles was conducted. The results for the rectus femoris muscle indicated that the sitting position, with rectus femoris acting over two joints, was not the best position for assessing the fatigue of this muscle. Further explanation of the results for rectus femons are presented in Appendix Three.  The slope of the median frequency for vastus lateralis decreased at two hours and then increased up to 20 hours post-exercise but the median frequency for vastus medialis did not change significantly over time (Table 7). The lack of significance for vastus medialis was most likely due to the small differences between time periods and the variability in the responses (Figure 9).  Roy (24) has commented that there is renewed interest in EMG spectral measurements for evaluating muscle fatigue due to the technical advances of digital systems and computer memory and processing speed.  He reports that it has been used in  ergonomics to assess muscle fatigue in occupational tasks and to assess muscle impairment in low back pain and neuromuscular disorders. However, he still considers it in the developmental stage. In this study there was a statistically significant difference over time in VL only. The decline in the slope of the median frequency of vastus lateralis at two hours post-exercise suggested that the greatest fatigue occurred in the muscle at this time but the difference between the pre-test and two hours post-exercise was not statistically significant (Figure 9). (See Appendix Three for further discussion.)  Muscle fatigue has been reported by others (21, 22) immediately following eccentric exercise and the pattern of response of VL in this study is similar to those reports.  80  Newham and colleagues (21) reported recovery from fatigue by 24 hours while Jones and colleagues (22) reported three to four days for recovery. In this study VL appeared to have recovered from fatigue by 20 hours post-exercise.  The significant negative correlations between eccentric torque and the median frequency of VL and the median frequency of VM at two hours and 20 hours post-exercise (Table 11) indicated that those subjects with the greatest eccentric torque were those who had the greatest decline in the slopes of the median frequencies of VL and/or VM, and vice versa. The greatest decline in the slope of the median frequency was equivalent to the greatest fatigue of the muscle.  The relationship that was expected was a positive  correlation. In order to understand these relationships, first the data was plotted and secondly, the slopes of the median frequencies of VL and VM were compared to the declining force of the quadriceps muscles during the 60 second isometric contraction of the power spectrum analysis.  From the plotted data it was obvious that an outlier existed. Figure 10 is an example of the plot of the correlation between eccentric torque and the slope of the median frequency of VL at two hours post-exercise. This same outlier was present in the three significant correlations between eccentric torque and the slopes of the median frequencies. When the relationships were examined without the outlier none of the correlations were significant  -  all three were equal to or less than r= -0.30.  Further examination of the slopes of the median frequencies of VL and VM and the slope of the declining force of the quadriceps muscles during the 60 second isometric contraction revealed a similar relationship as the one with eccentric torque. A single outlier (the same subject) transformed the lack of a relationship in the data into a significant linear  81  Figure 10. The Relationship Between Eccentric Torque and Vastus Lateralis Median Frequency at Two Hours Post-Exercise. TOR2 = eccentric torque, measured in Newton-meters. VL2 = the slope of the median frequency of the vastus lateralis muscle. The responses for each of the ten subjects are illustrated.  82  Figure 10  150  .  I  T 0 R 2  .  I  50 I  0  I  -0.6  -0.5  I  -0.4  -0.3 VLZ  -0.2  -0.1  83  relationship. Without this outlier there was no pattern in the data from the other nine subjects.  These two tests of muscle activity were different in that eccentric torque measured the force generating capability of the muscle during four maximal repetitions through 60 degrees of ROM at a velocity of 30 degrees/second. The power spectrum analysis assessed muscle fatigue, the progressive impairment of the force-generating capacity of the muscle (24), which involved a 60 second isometric contraction at 90 degrees of knee flexion.  The lack of a correlation between eccentric torque and the power spectrum analysis indicates that other central or peripheral factors may be contributing to the initial loss of strength in the muscle. Peripheral factors may include damage to the sarcomere (19), or loss of intracellular Ca homeostasis (32), or damage to the connective tissue (16), but the pattern of response of VL suggests that muscle fatigue should not be eliminated as one of the factors contributing to the decline in eccentric torque.  Another significant correlation between the slope of the median frequency of VL and intensity of soreness (r=-0.71) was found at four hours post-exercise (Table 11). It appeared that those subjects with the highest intensity of soreness score had the most fatigue in VL. However, once again, a single outlier was affecting the correlation. The outlier in this correlation represented a different subject but without this data point the linear relationship was not significant (r=-0.46). Further study of the relationships among the power spectrum analysis and the other dependent variables might benefit from larger subject numbers to either confirm or refute these results.  84  Conclusions  The significant differences in the presence of Tc-99m WBC between the sore, exercised muscle and the contra-lateral non-exercised muscle shows that leukocytes were present in the muscle in the first 24 hours following eccentric exercise. The substantial presence of Tc-99m WBC in the antero-distal aspect of the thigh at two hours postexercise, which remained at a high level for 24 hours, suggests that there may be greater damage to the muscle in this region due to increased stess at the muscle/tendon junction.  All of the dependent variables were significant over time except VM median spectral frequency and the presence of Tc-99m WBC in the antero-distal ROT. Intensity of soreness and unpleasantness were highest at 24 hours post-exercise while the loss of ROM was greatest at 24 hours post-exercise. The presence of Tc-99m WBC in the anterior, medial and lateral ROI increased up to 20 and 24 hours post-exercise but data collection did not proceed beyond 24 hours. The median frequency of vastus lateralis declined at two hours post-exercise.  In this study data were collected between the end of the exercise stimulus and 24 hours, at two, four and 20 hours post-exercise. By doing so a biphasic pattern in the response of eccentric torque was illustrated in humans that has only been reported previously in animal studies. Eccentric torque declined at 0 hour, recovered, and then declined again at 20 to 24 hours post-exercise. This pattern of response, which was different from intensity of soreness, unpleasantness, ROM and fatigue in this study, provides some explanation as to why measures of muscle strength have not correlated with other outcome measures of exercise-induced muscle damage in the muscle injury literature. The biphasic response of eccentric torque also supports the suggestion that there is more than one mechanism underlying exercise-induced muscle injury.  85  The only significant correlations in this study that had a linear relationship and were representative of the data, were between ROM and unpleasantness at 20 hours postexercise, and between intensity of soreness and unpleasantness at the pre-test and 72 hours post-exercise. The lack of a relationship between eccentric torque and either VL median frequency or VM median frequency in nine out of ten subjects, suggests that other factors, in addition to muscle fatigue, may be contributing to the decline in eccentric torque after the exercise stimulus.  86  References  1.  Smith L. Acute inflammation: The underlying mechanism in delayed onset muscle  soreness? Medicine and Science in Sports and Exercise 1991;23:542-551.  2.  Armstrong RB, Ogilvie RW, Schwane JA. Eccentric exercise-induced injury to rat  skeletal muscle. Journal of Applied Physiology 1983; 54:80-93.  3.  Zerba E, Komorowski TE, Faulkner JA. Free radical injury to skeletal muscles of  young, adult and old mice. American Journal of Physiology 1990;258: c429- c435.  4.  Ebbeling CB, Clarkson PM. Exercise-induced muscle damage and adaptation.  Sports Medicine 1989;7: 207-234.  5.  Stauber WT. Exercise and Sport Science Reviews.Baltimore: Williams and  Wilkins, 1989(vol 17): 157-185. Eccentric action of muscles: physiology, injury, and adaptation.  6.  Newham DJ, Jones DA, Ghosh G, Aurora P. Muscle fatigue and pain after  eccentric contractions at long and short length. Clinical Science 1988:74:553-557.  7.  Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric  exercise in man. International Journal of Sports Medicine 1983;4: 170- 176.  8.  Yonker RA, Webster EM, Edwards NL, et al. Technetium pyrophosphate muscle  scans in inflammatory muscle disease. British Journal of Rheumatology 1987;26:267-269.  87  9.  Jones DA, Newham DJ, Round JM, Toifree SEJ. Experimental human muscle  damage: morphological changes in relation to other indices of damage. Journal of Physiology 1986;375:435-44&  10.  Newham DJ, Jones DA, Toifree SE, Edwards RH. Skeletal muscle damage: a  study of isotope uptake, enzyme efflux and pain after stepping. European Journal of Applied Physiology 1986;55(1): 106-112.  11.  Willerson if, Parkey RW, Bonte FJ, Lewis SE, Corbett J, Buja LM.  Pathophysiologic considerations and clinicopathological correlates of technetium-99m stamious pyrophosphate myocardial scintigraphy. Seminars in Nuclear Medicine 1980;10(1):54-69.  12.  Kowalsky RJ, Perry JR. Radiopharmaceuticals in Nuclear Medicine  Practice.Norwalk, CT: Appleton and Lange, 1987  13.  Evans WJ, Cannon JO. Exercise and Sports Science Reviews.Baltimore: Williams  and Wilkins, 1991(vol. 19):99-125. The metabolic effects of exercise-induced muscle damage.  14.  Smith LL, McCammon M, Smith S, Chamness M, Israil RG, O’Brien KR White  blood cell response to uphill walking and downhill jogging at similar metabolic loads. European Journal of Applied Physiology 1989;58:833-837.  15.  Clarkson PM, Tremblay I. Exercise-induced muscle damage, repair, and adaptation  in humans. Journal of Applied Physiology 1988;65: 1-6.  88  16.  Jones DA, Newham DJ, Clarkson PM. Skeletal muscle stiffness and pain  following eccentric exercise of the elbow flexors. Pain 198730:233-242.  17.  Howell JN, Chila AG, Ford G, David D, Gates T. An electromyographic study of  elbow motion during postexercise muscle soreness. Journal of Applied Physiology 1985;58(5): 1713-1718.  18.  Fridén J, Sfakianos PN, Hargens AR, Akeson WH. Residual muscular swelling  after repetitive eccentric contractions. Journal of Orthopaedic Research 1988;6:493-498.  19.  Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during  contractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12): 911921.  20.  Davies CTM, White MJ. Muscle weakness following eccentric work in man.  Pflugers Archiv 1981 ;392: 168- 171.  21.  Newham DJ, Mills KR, Quigley BM, Edwards RHT. Pain and fatigue after  concentric and eccentric contractions. Clinical Science 1983 ;64: 55-62.  22.  Jones DA, Newham DJ, Torgan C. Mechanical influences on long-lasting human  muscle fatigue and delayed-onset pain. Journal of Physiology 1989;412:415-427.  23.  Frascarelli M, Rocchi L, Feola I. EMG computerized analysis of localized fatigue in  duchenne muscular dystrophy. Muscle and Nerve 1988;1 1:757-76 1.  89  24.  Roy SH. Combined use of surface electromyography and P3 1-NMR spectroscopy  for the study of muscle disorders. Physical Therapy 1993 ;73(12):892-901.  25.  Tarkka TM. Power spectrum of electromyography in arm and leg muscles during  isometric contractions and fatigue. Journal of Sports Medicine 1984;24: 189-194.  26.  Zwarts MJ, Van Weerden TW, Haenen HTM. Relationship between average  muscle fibre conduction velocity and emg power spectra during isometric contraction, recovery and applied ischemia. European Journal of Applied Physiology 1987;56:212-216.  27.  Delagi EF, Perotto A, lazetti J, Morrison D. Anatomic Guide for the  Electromyographer.Springfield, IL: C. Thomas, 1975  28.  Rothstein JM, Miller PJ, Roettger RF. Goniometnc reliability in a clinical setting.  Physical Therapy 1983;63(1O): 161 1-1615.  29.  Glass GV, Hopkins KD. Statistical Methods in Education and Psychology. (2 ed.)  Boston: Allyn and Bacon, 1984  30.  Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscle  damage and rapid adaptation. Medicine and Science in Sports and Exercise 1992 ;24(5):5 12-520.  31.  Rodenburg JB, Bar PR, de Boer RW. Relations between muscle soreness and  biochemical and functional outcomes of eccentric exercise. Journal of Applied Physiology 1993 ;74(6): 2976-2983.  90  32.  Armstrong RB. Initial events in exercise-induced muscular injury. Medicine and  Science in Sports and Exercise 1990;22(4):429-435.  91  CHAPTER THREE CONCLUDING REMARKS Collecting data after the exercise stimulus and before 24 hours revealed the biphasic response of eccentric torque following eccentric exercise which has not been reported before in humans. Eccentric torque decreased at 0 hour, recovered, and then decreased again at 20 to 24 hours post-exercise. This response may explain why eccentric torque does not correlate with other outcome measures. It would also support the hypothesis that more than one mechanism underlies exercise-induced injury in muscle.  Two of the proposed mechanisms associated with loss of muscle strength following eccentric exercise are muscle fatigue immediately after the exercise stimulus and a biochemical response to the phagocytic activity at 24 hours post-exercise (1). The results of this study indicate that fatigue, measured as slowing of the action potential conduction velocity, may be related to the loss of strength, in that the slope of the median frequency of vastus lateralis (VL) declined at two hours post-exercise but it was not significantly correlated with eccentric torque in nine out of ten subjects. Muscle fatigue may be more important to the loss of muscle strength earlier than two hours in the post-exercise period. The difference between the means of the median frequency of VL at the pre-test and two hours post-exercise in the study outlined in Chapter Two was small (0.04), while the difference between the means at the pre-test and 0 hour in the pilot study outlined in Appendix Three was larger (0.13 1). This is an area of further study, which would also benefit from a correlation analysis of torque and fatigue in the first few hours post-exercise.  Factors other than fatigue may also contribute to the loss of force immediately after the eccentric exercise. Damage to the sarcomere may physically limit the ability of the  92  muscle to produce force (1) or loss of calcium homeostasis may contribute further to the initial injury (2) and it may affect the functioning of the contractile unit (3).  In nine out of ten subjects who participated in the study outlined in Chapter Two, the region of interest (ROT) with the greatest presence of technetium-99m white blood cells (Tc-99m WBC) at two hours post-exercise was the antero-distal ROT. It appears that this ROT represented the muscle/tendon junction of the quadriceps muscle and that the leukocyte response to the injury was earlier than in the other ROT. The presence of Tc-99m WBC in the other ROT gradually increased up to 20 and 24 hours post-exercise while the presence of Tc-99m WBC in the antero-distal ROI was high at two hours post-exercise and remained high over the 24 hours. These results indicate that the stresses to the muscle/tendon junction are immediate and ongoing over 24 hours and different in timing to the response to the injury within the muscle fibre. This may suggest tissue specificity as a result of exercise-induced muscle injury. Although it has been suggested that there is both muscle fibre and connective tissue damage following eccentric exercise (4), it is not known in what proportion they occur or how they relate to each other or how injury to each tissue affects the symptoms of exercise-induced muscle injury.  In the study outlined in Chapter Two intensity of soreness and unpleasantness reached their highest levels at 24 hours post-exercise while range of motion (ROM) had the greatest loss at 24 hours and eccentric torque declined for the second time at 24 hours post exercise. Tt would be preliminary to conclude that the presence of Tc-99m WBC was related to the changes in the other outcome measures because Tc-99m WBC data was not collected beyond 24 hours and the Tc-99m WBC data was not correlated with the other outcome measures. But the results indicate directions for future study.  93  In both studies the Descriptor Differential Scale (DDS), which assessed more than one dimension of discomfort, was utilized. Using the DDS, the subjects’ perception of intensity of soreness as well as their perception of unpleasantness of soreness affective or emotional response to discomfort  -  -  the  was examined. The results suggest that  although the affective domain was not a primary experience for the subjects, in that the highest unpleasantness response was lower than that of the intensity of soreness, the two dimensions were related. Taking into consideration the different sample sizes, there were only two test times (at 20 and 24 hours in Chapter Two) when the two measures were not significantly correlated.  The results of the statistical analysis of creatine phosphokinase (CPK) indicated that there were no significant differences among the measures even though there was a large difference in the means at 96 hours after the exercise stimulus. The power of the analysis was 0.478 suggesting a 52% probability of a Type II error, or the statistical conclusion that there is no difference when in reality there is a difference. The pattern of the responses was similar to what has previously been reported in the literature and there were significant correlations with intensity of soreness and unpleasantness. The additional information contributed by this examination of patterns and relationships, together with the power analysis, cautions the investigator not to dismiss findings because the outcomes were not statistically significant. In this case, the lack of significance was most likely due to the characteristic inter-subject variability of CPK.  The cause of delayed muscle soreness following eccentric exercise is not known. The unpleasantness response of the DDS was significantly correlated with CPK at four and seven days post-exercise and ROM at 20 hours post-exercise. There were also significant correlations between intensity of soreness and CPK at four and seven days post-exercise.  94  The relationship between loss of ROM and unpleasantness suggests that pressure from swelling stimulates sensory nerve endings. However, the relationship between CPK and either intensity of soreness or unpleasantness does not appear as obvious, unless the relationship is reflecting the time it takes for CPK to reach the circulation through the interstitium. The greater the amount of swelling and damage to the connective tissue (5), and thus discomfort, the longer it takes for CPK to reach the circulation.  As investigators attempt to understand the mechanisms of muscle injury, the cause of muscle soreness, and the subsequent repair of muscle tissue, greater knowledge is obtained about the muscle at many levels. This knowledge is also being applied to the study of muscle diseases. As well, investigations concerning the morphological and ultrastructural changes in the muscle in response to eccentric exercise and overload are providing information about the way the contractile component functions to produce eccentric activity. As the mechanism by which muscle generates force is still mostly based on theory (6) these are important contributions to the study of muscle.  Future research from these studies may focus on radionuclide labelling of specific inflammatory cells such as neutrophils, macrophages and lymphocytes in order to better document the time course of the inflammatory process in muscle after eccentric exercise. These techniques may then be applied to the study of the response of debilitated muscle to exercise after prolonged immobilization.  95  References  1.  Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during  contractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1921.  2.  Armstrong RB. Initial events in exercise-induced muscular injury. Medicine and  Science in Sports and Exercise 1990 ;22(4):429-435.  3.  Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscle  damage and rapid adaptation. Medicine and Science in Sports and Exercise 1992 ;24(5) :512-520.  4.  Stauber WT, Clarkson PM, Fritz VK, Evans WJ. Extracellular matrix disruption  and pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.  5.  Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes after  strenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine 1992;119(2): 183  6.  -  188.  Chapman AE. Exercise and Sport Science Reviews.Baltimore: Williams and  Wilkins, 1985:443-501. The mechanical properties of human muscle.  96  APPENDIX ONE  Review of the literature Skeletal muscle is one of the most adaptable tissues in the body (1). Whether the activity level increases or decreases, the muscle responds continuously to the changes in its internal and external environment (2). It was commonly believed, until recently, that skeletal muscle fibres were unable to repair themselves after injury or disease, but now the ability of skeletal muscle to regenerate after injury is recognized (2) and the similarity of the process to the embryonic development of muscle has been observed (1).  Following serious injuries or prolonged immobilization, recovery of complete endurance and strength is often a slow and arduous task. This may be related to the ability of the debilitated skeletal muscle to respond to exercise, however, much of it may be related to a trial and error approach to exercise progression based on the patient’s feedback. If the training intensity is too small, progression will be much slower than necessary. However, if the training intensity is too severe, fatigue may persist and there may be alterations in the muscle consisting of cellular and structural damage.  Although some cell death and turnover may be an important component of the process optimizing the adaptation of skeletal muscle’s response to training, significant lethal cell damage may severely hinder recovery. The ability of skeletal muscle to regenerate after damage has been well established in mammalian species (3), however, it has been estimated that repair of damaged muscle may take as long as twelve weeks (4). Healthy individuals who had run a marathon demonstrated ultrastructural changes in their gastrocnemius indicative of degeneration and regeneration of skeletal muscle eight and ten  97  weeks following the race (5). From animal studies, it would appear that exercise facilitates alignment of normal architecture and optimal metabolism in muscle (2, 6).  Delayed onset muscle soreness (DOMS) is a sensation of discomfort associated with movement or palpation usually felt in skeletal muscle 24 to 72 hours following unaccustomed muscular exertion (7). Four major hypotheses have been put forth as being the causative factor of DOMS: high tension per unit area of muscle resulting in structural damage, increased metabolism resulting in the accumulation of waste products, increased temperature that causes structural damage, and altered neural control resulting in muscle spasm (7). Structural damage from high tension is the hypothesis that has received the most support to date. As early as 1902, Hough (8) described two distinct types of pain associated with exercise: pain which accompanies high intensity exercise likely related to metabolic or circulatory factors, and pain arising after the exercise (which may be caused by breaking of adhesions formed during the repair process resulting from rupture of contractile and connective tissue elements in the muscle).  The muscle activity which causes the most soreness and the most damage is eccentric activity (9, 10). During eccentric activity, the force developed is approximately twice that developed during isometric contractions.  The lowest forces are developed during  concentric contractions. The total number of attached cross bridges in a strongly bound state during eccentric activity is only about 10% greater than during an isometric contraction (11). Therefore, it seems probable that the mechanism of injury from eccentric exercise is due to the increased tension per individual cross bridge (11) causing mechanical disruption of the ultrastructural elements within the muscle fibres such as the Z-band and contractile filaments (12). Stauber and colleagues (13) have suggested that DOMS is due to a complex set of reactions involving disruption of the muscle fibre and connective tissue. The specific mechanism leading to DOMS, however, is still poorly understood.  98  Muscle soreness, or DOMS, is one of the characteristics of contraction-induced or exercise-induced muscle damage. Other characteristics include swelling, loss of muscle strength, and changes in the biochemical markers of the muscl&s integrity (14). Smith (15) has suggested that the sensation of muscle soreness, swelling and loss of function are also associated with the acute inllammatoiy response. This theory was initially proposed in the 1970’s but at that time was not supported by research findings. Similar to Stauber and colleagues (13), Smith (15) believes that mechanical disruption to the muscle fibre and connective tissue is a result of the unaccustomed eccentric exercise, but she goes on to point out that within a few hours of the injury the acute inflammatory response begins. First white blood cells migrate to the area, including neutrophils in the first few hours and monocytes 6-12 hours after the neutrophils.  When the monocytes enter the tissue  compartment from the blood, they mature into macrophages. The macrophages are at their greatest numbers at 48 hours. Their function is to remove the dead tissue but they are also important in the healing process. Smith (15) suggests that the presence of macrophages may be responsible for the synthesis of prostaglandins which may be related to the sensation of soreness at 48 hours.  It is not known whether decreased ROM reflects shortening of the contractile mechanism (16), increased edema within the connective tissue resulting in increased pressure (17), or injury to the connective tissue (14) or a combination of all three. Evidence of damage to the connective tissue is limited (10).  Abraham (18) reported  elevated hydroxyproline, a component of collagen, after exercise which included eccentric activity. However, few other studies have confirmed his results. Fritz and Stauber (19) showed noticeable histological variation by 24 hours in the proteoglycan component of the extracellular matrix (ECM) of rat muscle following eccentric activity. They suggested that structural disruption of the proteoglycan component may result in attraction of water within  99  the ECM as part of an osmotic force that leads to fluid accumulation. Clarkson and colleagues (14) have reported that changes in the circumference of the upper arm after eccentric exercise of the elbow flexors peaked at five days post-exercise while changes in the relaxed elbow angle (the angle at the elbow when the arm hangs freely by the side) were greatest at three days. Therefore, they believe that swelling and changes in ROM are not related. As Stauber (10) has commented, further research is necessary to support the hypothesis of connective tissue damage after eccentric exercise.  After fatiguing eccentric exercise, there is an immediate decrease in maximal force production which has been observed as early as one hour after the exercise (12, 20, 21). Newham and colleagues (22) reported that strength returned to the pre-exercise levels within 24 hours but others have found that it has taken as long as a week (21) or more (11). Assessing the relationship between development of soreness and the loss of muscle strength suggests that there is little or no relationship between the two (16). Thus, it seems unlikely that muscle soreness contributes significantly to the loss of muscle force.  It is not known if the initial decline in strength is due to fatigue or muscle injury. Clarkson and colleagues (14) have proposed that the immediate loss of strength may be due to overstretched sarcomeres in which the overlap between actin and myosin filaments would be reduced, thereby affecting force production. The fact that Newham and colleagues (23) have found greater strength losses after eccentric exercise at a long muscle length compared to a short muscle length lends support to this hypothesis.  Faulkner and colleagues (11) have reported a biphasic response in the muscle force in animals after eccentric exercise. They believe that the initial decline in force may be a function of mechanical injury and fatigue, especially where the subjects have just completed an exhaustive eccentric protocol. There is supporting evidence in the literature of  100  myofibrillar disruption at the level of the Z-line after eccentric exercise. Armstrong and colleagues (24) reported disruptions in the striation pattern of slow twitch fibres of the extensor muscles in rats immediately following downhill running. Friden and colleagues (25) identified disorganization of the Z-line in human soleus muscle three days after downhill running.  Newham and colleagues (26) showed that extensive sarcomeric  disruptions had occurred in human quadriceps muscles immediately after eccentric contractions. Areas of damage have been observed immediately after exercise with continuing disruption to the Z-line over the next few days (27).  Faulkner and colleagues (11) go on to suggest that a secondary injury occurs to the muscle and that it is a biochemical one as a result of the phagocytic activity at the site of the original damage. Neutrophils and monocytes release oxygen radicals, potentially causing further damage to the muscle.  When monocytes enter the tissue they mature into  macrophages (15). Animal studies have provided evidence of the presence of macrophages in the injured muscle after eccentric exercise (24) but human morphological studies have not provided firm evidence of the acute phase response in the exercised muscle within 48 hours of eccentric exercise (25, 28). Evans and Cannon (29) believe that the absence of morphological evidence in human studies is mostly due to sampling procedures which miss the region of injury or timing that is later than the acute phase response.  Some investigators have assessed products released in the urine, blood or plasma from the breakdown of connective tissue or muscle, or the increased permeability of the muscle fibre.  Of these techniques, most of the studies to date have investigated the  relationship between DOMS and muscle products such as creatine phosphokinase (CPK), lactate dehydrogenase or myoglobin (7, 18, 28, 30-35).  One of the most commonly  studied serum proteins is the response of CPK after exercise (36). CPK, a marker for some muscle diseases (14), has been shown to increase after eccentric exercise but it does  101  not have the same time course as the perception of soreness (13). It usually begins to increase by 24 hours after the eccentric exercise and reaches its highest values three to six days post-exercise (36).  Other muscle proteins, such as lactate dehydrogenase and  myoglobin, show a similar delay in their increase in the blood after eccentric exercise (14).  CPK is a key enzyme in the ADP ATP transformation (37). It is released from the -  muscle tissue into the interstitium and then it travels to the circulation via the lymphatic system (16). It is not known what mechanism mediates the release of CPK from the tissue. Evans and Cannon (29) have stated that the post-exercise CPK response is a manifestation of muscle damage but not a direct indicator of it.  An early investigation by Abraham (18) examined a marker of connective tissue damage, the appearance of urine hydroxyproline (OHP). Further support of the importance of connective tissue damage contribution to muscle soreness and poor performance comes from a more recent study where evidence of the disruption of extracellular matrix occurred at the same time as an increase in muscle soreness (13). Essentially all OHP in vertebrate tissue is found in collagen, except for a small amount in elastin (38).  This unique  distribution of OHP makes it a useful label for studying the metabolism of collagen (38).  Other investigators have also reported on OHP after exercise. Seaman and lanuzzo (34) reported a lack of significant change in the hydroxyproline/creatimne (OHP/Cr) ratio and serum Ol-IP following 30 minutes of double leg extensions. Horswill and colleagues (39) also reported a lack of significant change in the OHP/Cr ratio and OHP excretion following three circuits of nine exercises on a Universal Gym. However, Murguia and  102  colleagues (40) reported a significant baseline increase in the plasma OHP values of NAVY SEAL candidates who later developed connective tissue injuries during their training program, compared to candidates without injury. In other words, those at risk for connective tissue injuries had elevated OHP levels at the beginning of training.  The most direct evidence of muscle damage is provided by ultrastructural and histological analyses of muscle damage. Friden and colleagues (12) have shown that there is broadening, streaming and sometimes total disruption of the Z-bands following eccentric exercise. They noted that these changes continued to increase up to three days after the insult but by six days the evidence of muscle damage had reduced. They have also noted changes in the sarcolemma (12). Some authors have suggested there may be an association between the damaged sarcolemma and increases in levels of calcium ions (22, 41). The levels of calcium ions are important in both inducing muscle damage and in the repair process but Ebbeling and Clarlcson (16) have cautioned that much more investigation is needed to understand the effect of altered levels of calcium ions on the muscle. Stauber and colleagues (13) observed separation of the extracellular matrix, mast cell degranulation, and increased plasma constituents in the extracellular space in biopsies taken at 48 hours. They suggested that the muscle soreness also seen at 48 hours is a result of the inflammatory response which they believe is in response to the extracellular disruption. However, in their study biopsies were taken at only one time period (48 hours).  Although there has been considerable success in producing an exercise model which reproduces the contraction-induced injury in skeletal muscle, the mechanism of the injury is still not known. Most researchers believe that the initiating event is a mechanical one but the possibility of a metabolic component contributing to the injury has also been suggested (11). Faulkner and colleagues (11) have suggested that during lengthening of the muscle, some sarcomeres maintain their length while others are stretched beyond overlap and  103  injured. Lieber and Fnden (42) have proposed that the cytoskeleton of muscle is the first structure to yield after eccentric activity, followed by myofibrillar disruption of the A-band and the Z-band. They have also suggested that, because of the damage to the Type II fibres in human muscle, fibre oxidative capacity might be an important factor in determining the extent of fibre damage following eccentric exercise (43). Perhaps the Type II fibres fatigue early in the exercise; they are unable to regenerate ATP and subsequently enter a “stiffness” state. These stiff fibres are then stretched and damaged during the eccentric activity. Clarkson and colleagues (14) have also proposed that there may be stiffness in the cross bridge but they believe that it occurs for a few days after the exercise as a result of increased intracellular calcium (Ca++). Armstrong (41) has also proposed that as a result of injury to the sarcolemma there is a loss of Ca++ homeostasis in the muscle. This stage of events occurs before the inflammatory cellular response and begins the degradative process through activation of phospholipase A2 in the cell membrane and the subsequent leukotriene and prostaglandin cascades.  The challenge is in determining the underlying mechanism of injury in an intact species. Exercise is described as promoting strengthening of the connective tissue harness of muscle and through this mechanism is thought to be a preventive therapy for injury to these structures. However, if the exercise stimulus exceeds the ability of the skeletal muscle tissue to respond then DOMS, and muscle fibre and connective tissue damage will occur. An optimal training level is achieved when the intensity of the exercise program stimulates new muscle tissue growth but minimizes excessive muscle damage. Thus, the objectives of these studies were to characterize the time course and relationships of outcome measures of muscle injury after eccentric exercise and to examine the presence of inflammatory cells in the exercised muscle.  104  References  1.  Lieber RL Skeletal Muscle Structure and Function.Baltimore: Williams and  Wilkins, 1992  2.  Carison BM. Regeneration of entire skeletal muscles. Federation Proceedings  1986;45: 1456-1460.  3.  Carlson BM, Faulkner JA. The regeneration of skeletal muscle fibres following  injury: a review. Medicine and Science in Sports and Exercise 1983;15: 187- 198.  4.  Evans WJ. Exercise-induced skeletal muscle damage. The Physician and  Sportsmedicine 1987; 15( 1)(Jan) :88-100.  5.  Warholl MJ, Siegal AJ, Evans WJ, Silverman LM. Skeletal muscle injury and  repair in marathon runners after competition. American Journal of Pathology 1985;1 18:33 1-339.  6.  White TP. Adaptations of skeletal muscle grafts to chronic changes of physical  activity. Federation Proceedings 1986;45: 1470-1473.  7.  Armstrong RB. Mechanisms of exercise-induced delayed onset muscular soreness:  a brief review. Medicine and Science in Sports and Exercise 1984;16(6):529-538.  8.  Rough T. Ergographic studies in neuro-muscular fatigue. American Journal of  Physiology 1902;7:76-92.  105  9.  McCully KK, Faulkner JA. Injury to skeletal muscle fibers of mice following  lengthening contractions. Journal of Applied Physiology 1985;59( 1): 119-126.  10.  Stauber WT. Exercise and Sport Science Reviews.Baltimore: Williams and  Wilkins, 1989(vol 17): 157-185. Eccentric action of muscles: physiology, injury, and adaptation.  11.  Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during  contractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1921.  12.  Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric  exercise in man. International Journal of Sports Medicine 1983;4: 170- 176.  13.  Stauber WT, Clarkson PM, Fritz VK, Evans Wi. Extracellular matrix disruption  and pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.  14.  Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscle  damage and rapid adaptation. Medicine and Science in Sports and Exercise 1992 ;24(5) :512-520.  15.  Smith L. Acute inflammation: The underlying mechanism in delayed onset muscle  soreness? Medicine and Science in Sports and Exercise 1991;23:542-551.  16.  Ebbeling CB, Clarkson PM. Exercise-induced muscle damage and adaptation.  Sports Medicine 1989;7:207-234.  106  17.  Jones DA, Newham DJ, Clarkson PM. Skeletal muscle stiffness and pain  following eccentric exercise of the elbow flexors. Pain 1987;30:233-242.  18.  Abraham WM. Factors in delayed muscle soreness. Medicine and Science in  Sports 1977;9:11-20.  19.  Fritz VK, Stauber WT. Characterization of muscles injured by forced lengthening.  II. Proteoglycans. Medicine and Science in Sports and Exercise 1988;20(4):354-361.  20.  Clarkson PM, Tremblay I. Exercise-induced muscle damage, repair, and adaptation  in humans. Journal of Applied Physiology 1988;65: 1-6.  21.  Newham DJ, Jones DA, Clarkson PM. Repeated high force eccentric exercise:  Effects on muscle pain and damage. Journal of Applied Physiology 1987;63: 1381-1386.  22.  Newham DJ, Mills KR, Quigley BM, Edwards RHT. Pain and fatigue after  concentric and eccentric contractions. Clinical Science 1983 ;64:55-62.  23.  Newham DJ, Jones DA, Ghosh G, Aurora P. Muscle fatigue and pain after  eccentric contractions at long and short length. Clinical Science 1988;74:553-557.  24.  Armstrong RB, Ogilvie RW, Schwane JA. Eccentric exercise-induced injury to rat  skeletal muscle. Journal of Applied Physiology 1983; 54:80-93.  25.  Fndén J, SjOstrOm M, Ekblom B. A morphological study of delayed muscle  soreness. Experientia 1981 37:506-507.  107  26.  Newharn DJ, McPhail G, Mills KR, Edwards RHT. Ultrastructural changes after  concentric and eccentric contractions of human muscle. Journal of The Neurological Sciences 1983;61: 109-122.  27.  Newham DJ. The consequences of eccentric contractions and their relationship to  delayed onset muscle pain. European Journal of Applied Physiology 1988;57:353-359.  28.  Jones DA, Newham DJ, Round JM, Tolfree SET. Experimental human muscle  damage: morphological changes in relation to other indices of damage. Journal of Physiology 1986;375:435-448.  29.  Evans WJ, Cannon JG. The metabolic effects of exercise-induced muscle  damage.Baltimore: Williams and Wilkins, 1991:99-125. Exercise and Sports Science Reviews  30.  Byrnes WC, Clarkson PM, White JS, Hsieh SS, Frykman PN, Maughan RJ.  Delayed onset muscle soreness following repeated bouts of downhill running. Journal of Applied Physiology 1985 ;59(3):710-715.  31.  Clarkson PM, Byrnes WC, McCormick KM. Turcotte LP, White JS. Muscle  soreness and serum creatine kinase activity following isometric, eccentric and concentric exercise. International Journal of Sports Medicine 1986;7(3): 152-155.  32.  Newham DJ, Jones DA, Tolfree SE, Edwards RH. Skeletal muscle damage: a  study of isotope uptake, enzyme efflux and pain after stepping. European Journal of Applied Physiology 1986;55(1): 106-112.  108  33.  Schwane JA, Williams JS, Sloan JH. Effects of training on delayed muscle  soreness and serum creatine kinase activity after running. Medicine and Science in Sports and Exercise 1987; 19(6):584-590.  34.  Seaman R, lanuzzo CD. Benefits of short-term muscular training in reducing the  effects of muscular over-exertion. European Journal of Applied Physiology 1988 ;58(3):257-261.  35.  Thomas BD, Motley CP. Myoglobinemia and endurance exercise: A study of  twenty-five participants in a triathion competition. American Journal of Sports Medicine 1989;12(2): 113-119.  36.  Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes after  strenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine 1992;119(2): 183  37.  -  188.  Hortobágyi T, Denahan T. Variability in creatine kinase: methodological, exercise,  and clinically related factors. International Journal of Sports Medicine 1989; 10(2):69 80. -  38.  Prokop DJ, Kivirikko KI. Relationship of hydroxyproline excretion in urine to  collagen metabolism. Annals of Internal Medicine 1967;66(6): 1243 1266. -  39.  Horswill CA, Layman DK, Boileau RA, Williams BT, Massey BH. Excretion of 3-  methylhistidine and hydroxyproline following acute weight-training exercise. International Journal of Sports Medicine 1988;9(4):245-248.  109  40.  Murguia MJ, Vailas A, Mandelbaum B, eta!. Elevated plasma hydroxyproline A  possible risk factor associated with connective tissue injuries during overuse. American Journal of Sports Medicine 1988;16(6):660-664.  41.  Armstrong RB. Initial events in exercise-induced muscular injury. Medicine and  Science in Sports and Exercise 1990;22(4):429-435.  42.  Lieber RL, Friden J. Muscle damage is not a function of muscle force but active  muscle strain. Journal of Applied Physiology 1993 ;74(2):520-526.  43.  Fridén J, Lieber RL. Structural and mechanical basis of exercise-induced muscle  injury. Medicine and Science in Sports and Exercise 1992;24(5):521-530.  110  APPENDIX TWO  Descriptor Differential Scale (DDS)  I Instructions to the Subjects  Figures 11 and 12 are examples of the intensity of soreness scale and the unpleasantness scale respectively. Subjects scored their intensity of soreness and unpleasantness immediately following the test of eccentric torque, which was meant to be the reference activity for scoring the DDS. Subjects scored the DDS on a computer where the descriptors were generated onto the screen one at a time and in a randomized order. The subjects were instructed to think about their soreness relative to each anchoring descriptor. If the descriptor perfectly described their soreness, then they were instructed to mark underneath it; if their soreness was proportionally less than that described by the descriptor then they were to mark to the left of the descriptor; and if their soreness was proportionally more than that described by the descriptor they were to mark to the right of the descriptor (1).  II Calculations  The 12 descriptors were placed in order of increasing descriptor magnitude according to Gracely and Kwilosz (1). Each descriptor was scored from -10 to +10. The data for each scale was plotted with the descriptors on the X axis and the scores on the Y axis. In an attempt to represent the increasing magnitude of the descriptors, the intercept of the line of best fit through the 12 data points was used as the score of each scale. Figure 13 is an example of one subject’s soreness response at 24 hours post-exercise. The descriptors on the X axis are ranked in order of increasing magnitude. Figure 14 is an  111  Figure 11. Amount of Sensation. The 12 descriptors of the intensity of soreness scale are presented in randomized order. See text for explanation.  112  PART A Amount of Sensation  Figure 11  -  Rate your sensation in relation to each woni with a check mark.  Slightly Intense +  -  Mild +  -  Strong +  -  Faint +  -  Veiy Weak +  -  Very Intense +  -  Moderate +  -  Veiy Mild +  -  Weak +  -  Extremely Intense +  -  Barely Strong +  -  Intense -  +  113  Figure 12. Amount of Unpleasantness. The 12 descriptors of the unpleasantness scale are presented in randomized order. See text for explanation.  114  PART B Amount of Unpleasantness  Figure 12  -  Rate your unpleasantness in relation to each word with a check mark.  Very Unpleasant +  -  Very Distressing +  -  Slightly Unpleasant +  -  Unpleasant +  -  Slightly Intolerable +  -  Annoying ÷  -  Distressing +  -  Slightly Annoying +  -  Slightly Distressing +  -  Very Intolerable +  -  Intolerable +  -  Very Annoying +  115  Figure 13. Subject 1: Intensity of Soreness at 24 Hours Post-Exercise. On the y axis is the score for each descriptor for this subject. The descriptors are listed on the x axis in order of increasing magnitude.  EXTREMELY INTENSE  VERY INTENSE  INTENSE  STRONG  SLIGHTLY INTENSE  BARELY STRONG  MODERATE  MILD  VERY MILD  WEAK  VERY WEAK  FAINT  U’  0  Q  •  •  I  L.fl  .  I  •  Lfl  .  .  I  0 -S CD  I-a  117  Figure 14. Subject 1: Unpleasantness Response at 96 Hours Post-Exercise. The descriptions of the y axis and x axis are the same as Figure 13. Note that different words have been chosen as descriptors for this scale.  VERY INTOLERABLE  INTOLERABLE  VERY DISTRESSING  SLIGHTLY INTOLERABLE  VERY ANNOYING  ANNOYING SLIGHTLY DISTRESSING VERY UNPLEASANT DISTRESSING  UNPLEASANT  SLIGHTLY ANNOYING  SLIGHTLY UNPLEASANT  Ui  ‘‘I  .  Vi  •  .  -o  •  .  ,  CD  —s  Vi’  I-.  119  example of the same subject’s unpleasantness response at 96 hours post-exercise, with the descriptors in order of increasing magnitude on the X axis.  III Reliability and Validity  Purpose  The purpose of this pilot study was to assess the test-retest reliability and concurrent validity of the intensity of soreness and the unpleasantness scales of the DDS in subjects with DOMS. Gracely and Kwilosz (1) have previously reported the DDS to be reliable in a group of 91 dental patients.  Protocol  Seven subjects (four females and three males), ages 23 to 34 years, participated in a pilot study of the test-retest reliability of the DDS. Two of those subjects did not participate at 96 hours and 168 hours post-exercise. The subjects scored the DDS at the usual testing  times  -  pretest, and 24 hours, 48 hours, 96 hours and 168 hours following the eccentric  exercise (test 1). They returned one hour later, at each test session, and following four repetitions of the eccentric torque protocol, they scored the DDS again (test 2).  Eighteen subjects (14 females and four males) ages 20 to 52 years, scored their soreness according to the Visual Analogue Scale (VAS) in addition to the DDS. The VAS has been utilized in other studies of delayed onset muscle soreness (2). The DDS scores, over the five testing times, were correlated with the scores of the VAS to determine concurrent validity.  120  Data Analysis  Pearson product-moment correlation analyses were used to determine test-retest reliability between test 1 and test 2, and concurrent validity between the DDS (intensity of soreness and unpleasantness) and the VAS.  Results  Table 12 summarizes the test-retest reliability and the concurrent validity of the DDS. The test-retest reliability of the intensity of soreness ranged from 0.72 to 0.99 over the five test times. For the unpleasantness scale the correlations ranged from 0.64 to 0.98, except for the the pre-test day (r=0.58). On the pre-test day five out of seven subjects had the same scores (-10) on both test occasions. One subject had scores that were -9 at both times, and the other subject’s second score was within one point of the first score (-10). However, as these responses caused a clumping of the scores, and the Pearson r is a measure of linearity, the correlation was poor even though the scores were in fact very close over the two test times.  Richman and colleagues (3) have suggested a scheme to rank the correlation values  -  0.80 to 1.0 is “very reliable”; 0.60 to 0.79 is “moderately reliable” and 0.59 and less has “questionable reliability”. All of the intensity of soreness correlations for test-retest reliability would be considered very reliable except at 24 hours post-exercise, which would be considered moderately reliable. Two of the unpleasantness correlations would be termed very reliable and two would be considered moderately reliable. The pre-test correlation appears of questionable reliability but in fact the scores were very repeatable.  121  TABLE 12 PEARSON PRODUCT-MOMENT CORRELATION COEFFICIENTS FOR THE DESCRIPTOR DIFFERENTIAL SCALE Test-Retest Reliability for Intensity of Soreness Testl  Pre-test  24Hr  48Hr  96Hr  168Hr  n=7  n=7  n=7  n=5  n=5  Test2 Pre-test  0.92 0.72  24Hr  0.85  4SHr  0.80  96Hr  0.99  168Hr  Test-Retest Reliability for Unpleasantness Test 1 Pre-test  24Hr  48Hr  96Hr  168Hr  n=7  n=7  n=7  n=5  n=5  Test2 Pre-test  0.58 0.74  24Hr  0.97  48Hr  0.98  96Hr  0.64  168Hr  Concurrent Validity VAS Intensity of Soreness  0.84  Unpleasantness  0.63  122  The concurrent validity of the soreness scale of the DDS with the VAS for 18 subjects over two exercise durations and five test times was 0.84, while the validity of the unpleasantness scale with the VAS was 0.63. The higher correlation between the intensity of soreness scale and the VAS suggested that subjects scored the VAS relative to intensity of soreness.  These results suggest that the DDS is an objective measure of muscle soreness. They also substantiate the findings of Gracely and Kwilosz (1) that the DDS is a reliable instrument.  123  References  1.  Gracely RH, Kwilosz DM. The descriptor differential scale: applying  psychophysical principles to clinical pain assessment. Pain 198835:279-288.  2.  Bobbert MF, Hollander AP, Huijing PA. Factors in delayed onset muscular  soreness of man. Medicine and Science in Sports and Exercise 1986;18(1):75-81.  3.  Richman J, Makrides L, B P. Research methodology and applied statistics, Part 3:  Measurement procedures in research. Physiotherapy Canada 1980;32: 253-257.  124  APPENDIX THREE  Power Spectrum Analysis  I Calculations  The force and raw EMG signals were simultaneously collected by the computer for spectral analysis. The sampling rate was 500 Hz. Figure 15 illustrates two sections of the raw data from six to nine seconds and from 54 to 57 seconds respectively for one subject. As the subject fatigued and force declined, the EMG amplitude declined and there were more areas of the EMO that were relatively inactive (Figure 15 B) compared to the earlier signal (Figure 15 A).  Spectral analysis entailed partitioning the EMG data into overlapping four-second segments with each successive segment starting two seconds later than the previous segment. Spectral estimates of each four-second segment were then calculated through a Fast Fourier Transformation. Figure 16 is an example of the spectral frequency for the vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM) muscles for one foursecond segment. Lindstrom and Magnusson (1) have reported that as muscle fatigues the spectral frequency shifts to a lower frequency indicating a decrease in action potential conduction velocity. Other investigators suggest that although there is a relationship between spectrum analysis and conduction velocity, factors such as the rate of motor unit firing also contribute to the change in EMG signal frequency and thus, muscle fatigue (2).  The median frequency, which was calculated from the area under the curve, served as the measure by which muscle fatigue was calculated. The median frequency of each four second segment was plotted against time and a linear regression line was fitted to the 28  125  Figure 15. Force and Raw Electromyography (EMG) Signals. A. Recordings between 6 and 9 seconds. B. Recordings between 54 and 57 seconds. VL is the vastus lateralis muscle, RF is the rectus femons muscle, and VM is the vastus medialis muscle.  126  A  Figure 15  14000  I  12000  VL  10000 8000  RF  6000 ‘VM  4000 2000  0 Force  --  6  6:5  7.5  8  8.5  9  Time (seconds)  B 14.c)cX)  12000  —  I  k4k*M  ft  VL  100008000  RF  .6000VM  4000 2000  0-  54.5  56  Time (seconds)  Force 56.5  57  127  Figure 16. Typical Surface-EMG Power Spectrum. VL=vastus lateralis, RF=rectus femons, VM=vastus medialis. See text for explanation.  128  Figure 16  Typical Surface-EMO Power Spectrum low 4  4  I  10 4  U  t  4 .2  10  ft  \4\_%_4  ::::7:J::::::::::::::.:  ::::::::::::::::::::::::::::::  :::  ::::::Ib.JI5.Q.%,  :‘‘—“.“  .:::::::2;::::::::]::::::::::.:::::.:::::::::::::  10  VL RF  WI 106  1O  0  50  100  150  Frequency (Hz.)  200  250  129  Figure 17. Regression Lines for the Frequency Spectra. VL=vastus lateralis, RF=rectus femoris, VM=vastus medialis. “Half-Power” is the Median Frequency. See text for explanation.  130  Figure 17 100 yu  .  60 0 C) I_  4 Is  RF n II I --  VL, WI  I  U 0  04 nfl  20 IC  10  20  30 Time (seconds)  40  50  60  131  data points (Figure 17). The slope of the line was considered a quantitative measure of muscle fatigue. The steeper the negative slope of the line, the greater the muscle fatigue. Other investigators have sampled data at the beginning, middle and end of a contraction (3) or every 10 seconds throughout the contraction (4). By analyzing 60 seconds of data, in overlapping segments, 28 times, a more precise reflection of the changes in the frequency was achieved.  II Reliabffity and Validity  Purpose  The purpose of this pilot study was to determine if the power spectrum analysis is an objective measure of muscle fatigue.  Protocol  Six subjects (three females and three males), from 20 to 43 years, participated in a study of test-retest reliability and concurrent validity of the power spectrum analysis. The subjects repeated the protocol on two separate days not more than one week apart. The slope of the median frequency of each muscle was compared to the slope of isometric force to determine concurrent validity. Bigland-Ritchie (5) has stated that loss of force characterizes muscle fatigue.  Methods  The subjects were seated on the KinCom isokinetic dynamometer, with their hips at 80 degrees, their back supported and the pelvis stabilized on the bench. The centre of  132  rotation of the KinCom was positioned opposite the centre of the knee joint line. The resistance pad was positioned at a point on the lower leg that was 75% of the length of the fibula. The knee was positioned at 90 degrees of knee flexion.  The skin was cleaned with an alcohol swab and surface EMG electrodes (Medi Trace silver/silver chloride, circular, 1 cm radius) were placed over the motor points of VL, RF, and VM muscles according to Delagi and colleagues (6). The interelectrode spacing was 2.5 cm. A ground electrode was placed over the wrist. Electrodes were placed on the skin with electromedical gel between the skin and the electrodes. Subjects maintained a maximum isometric contraction of the knee extensors for 60 seconds. A ten-second submaximal isometric contraction preceded the one minute fatiguing contraction as a warm-up.  Data Analysis  Pearson product-moment correlation analyses were used to assess test-retest reliability of the slope of the median frequency for each muscle between day 1 and day 2, and concurrent validity between the slope of the median frequency of each muscle and the slope of the isometric force over the two test times. Isometric force, recorded every two seconds over the 60 seconds of the test, was plotted against time and a linear regression line was fitted to the data points. The slope of the line was the quantitative measure of the change in force.  Results  Table 13 summarizes the results of test-retest reliability and the concurrent validity of the power spectrum analysis. All of the correlations between day 1 and day 2 for the  133  TABLE 13 POWER SPECTRUM ANALYSIS:  PEARSON PRODUCT-MOMENT CORRELATION COEFHCIENTS Test-Retest Reliability VL1* VL2  VM1  RF1  0.99 0.90  RF2  0.98  VM2  *  VL1, VL2 are the Median Frequencies of Vastus Lateralis at Test 1 and Test 2  respectively. RF1, RF2 are the Median Frequencies of Rectus Femons at the same test times, and VM1, VM2 are the Median Frequencies of Vastus Medialis at the same test times. Concurrent Validity Force Median Frequency of Vastus Lateralis  0.88  Median Frequency of Rectus Femons  0.91  Median Frequency of Vastus Medialis  0.89  134  slopes of the median frequency of each of the muscles were greater than 0.90. The correlations between the slope of the median frequency of each muscle and the slope of the isometric force were all above 0.87.  These results suggest that the power spectrum analysis is a reliable and valid tool for quantifying muscle fatigue.  III Fatigue of the Rectus Femoris Muscle  Figure 18 is a graph of the mean slope of the median frequency of RF, plus and minus one standard deviation, for the ten subjects who participated in the study reported in Chapter Two. Contrary to the responses of VL and VM, the slope of the median frequency of RF increased following the eccentric exercise protocol. The results suggested that the muscle was not fatigued but this seemed unlikely after 300 eccentric contractions over 30 minutes. The response of RF was also different to the responses of the other two superficial quadriceps muscles even though all of the muscle are knee extensors. Further testing was conducted in an attempt to understand the response of RF.  As RF is a muscle that crosses both the hip and the knee joints, the position of the hip joint was changed to a neutral position. By lying subjects supine on the plinth, with the hip at 0 degrees, RF was isolated to working as a knee extensor at a longer muscle length.  Five female subjects (24 43 years), who were part of another study, participated -  in the same exercise stimulus and power spectrum analysis protocol as has been outlined in Chapter Two. Data was collected before the eccentric exercise (pre-test), immediately after the exercise protocol (0 hour) and at two hours post-exercise for the subjects. These subjects were postioned in sitting to confirm or reject the results for RF from the study  135  Figure 18. Power Spectrum Analysis: Rectus Femoris Median Frequency. Values are means+SD.  CD  U,  P  F1  [  U,  )  I  0’  p  I  I  0’  -  SLOPE OF THE MEDIAN FREQUENCY  I  c  P  -  Cr1  I.  C.)  137  outlined in Chapter Two. One subject did not participate in the data collection at two hours post-exercise. Table 14 summarizes the descriptive statistics and Figure 19 A illustrates the slopes of the median frequencies for the three quadriceps muscles VL, RF and VM. -  Similar to the early post-exercise responses of the subjects who participated in the study reported in Chapter Two, the slopes of the median frequencies of VL and VM declined over two hours post-exercise and the slope of the median frequency of RF increased.  Next, nine female subjects, 21 47 years, participated in the same protocol except -  that these subjects were postioned supine with the hip at 0 degrees. The knee was still positioned at 90 degrees of knee flexion. The instructions to these subjects were that they were not to lift their heads or their shoulders during the testing. Data were collected before the exercise stimulus and at 0 hour. Table 14 summarizes the descriptive statistics and Figure 19 B represents the slopes of the median frequencies for the three muscles (VL, RF, VM) from the pre-test toO hour. With a change in the position of the hip joint the slope of the median frequency of RF had decreased at 0 hour compared to the pre-test.  The results of these pilot studies suggest that the position of the hip joint influenced the power spectrum analysis of RF, but for VL and VM the pattern of the response was similar to that reported in Chapter Two. The most obvious difference, with the change in the hip position from 80 degrees of flexion to neutral, was that RF was working at a longer muscle length with the hip in neutral. The change in the hip joint position did not affect VL and VM as they only cross the knee joint.  According to the length-tension relationship of muscle, a shortened muscle with overlapping actin filaments within the sarcomeres, generates much less tension than at the  138  TABLE 14 POWER SPECTRUM ANALYSIS: DESCRIPTIVE STATISTICS OF THE MEDIAN FREQUENCIES OF THE QUADRICEPS MUSCLES WITH THE HIP AT 80 DEGREES FLEXION Test Times Pre-test  0 Hour  2 Hours  n=5  n=5  n=4  VastusLateralis  -0.115 (0.05)  -0.199 (0.13)  -0.161 (0.08)  Rectus Femons  -0.234 (0.13)  -0.180 (0.13)  -0.193 (0.06)  VastusMedjalis  -0.115 (0.11)  -0.163 (0.08)  -0.192 (0.11)  Data are means (SD).  WITH THE HIP AT 0 DEGREES FLEXION Test Times Pre-test  0 Hour  n=9  n=9  Vastus Lateralis  -0.127 (0.08)  -0.258 (0.09)  Rectus Femons  -0.194 (0.08)  -0.253 (0.07)  Vastus Medialis  -0.087 (0.07)  -0.254 (0.11)  Data are means (SD).  139  Figure 19. Slopes of the Median Frequencies of the Superficial Quadriceps Muscles. A. With the hip in 80 degrees of flexion. B. With the hip neutral. Values are means + SD. VL=vastus lateralis, RF=rectus femons, VM=vastus medialis.  liii!)  II  r  ++  C  C’,  -‘  0  XC Cd)  -‘  0  m  r.4  D  m  I  P -  -1  -  p I\j  -I  CD  p  caj  SLOPE OF THE MEDIAN FREQUENCY  -v CD  P  C_13 (rI  P (11  P (11  SLOPE OF THE MEDIAN FREQUENCY  C  5  CD  1  C  141  resting length of the muscle (7). The exact resting length of rectus femoris in vivo is not known, but with the hip at 80 degrees of flexion and the knee at 90 degrees of flexion the muscle may have been positioned in a relatively shortened position. If there was little tension development there would be little evidence of fatigue over 60 seconds. Lieber (7) has stated that muscle and joint relationships have not been thoroughly studied in humans. The biomechanics and force-generating properties of muscles in regard to fibre length, fibre and muscle area, tendon length and moment arms must be considered. In addition, the architecture of the muscle contributes to its tension-producing capacity (7). The arrangement of muscle fibres either enhances the strength producing capabilities or the excursion of the muscle.  Further investigation of the response of RF to fatiguing exercise is required. A research design in which subjects repeated the experiment twice with the order of the position of the hip randomly assigned, with hip position as the independent variable, and including both male and female subjects, would determine if hip position does influence the response of RF during power spectrum analysis. The other possibility is that subjects may have rotated the hip internally or externally in sitting, selectively recruiting VL and VM respectively, during the isometric knee extension. Strapping of the thigh to prevent internal and external rotation would eliminate this possible confounding variable.  IV Fatigue of the Vastus Lateralis and Vastus Medialis Muscles  The results of the post hoc testing of the slopes of the median frequencies of VL and VM in Chapter Two showed a significant difference only between four hours and 20 hours post-exercise for VL, when the muscle appeared to be recovering from fatigue.  142  There were no significant differences between the pre-test and two hours post-exercise for either muscle.  In this pilot study a paired t-test of the slopes of the median frequencies of the three muscles was used to analyze the differences between the pre-test and 0 hour for the nine subjects whose results are illustrated in Figure 19 B. These subjects were postioned with the hip in neutral. Table 15 summarizes the results of the paired t-test. For all of the muscles the slopes of the median frequencies significantly declined from the pre-test to 0 ). These results should be interpreted cautiously, however, because the t-test 5 hour (p.cO.O only analyzes the differences between the means (8).  Although others have suggested that the muscle’s recovery from fatigue may take one or more days (9, 10), Faulkner and colleagues (11) have proposed that most of the recovery takes place in the first three hours post-exercise. The results of these pilot studies suggest that their observations may be correct and that muscles quickly recover even from a strenuous exercise regime.  143  TABLE 15 POWER SPECTRUM ANALYSIS PAIRED T-TESTS Source  mean difference  Vastus Lateralis Median Frequency  (df, 8)  pre-test-Ohr Rectus Femoris Median Frequency pre-test-Ohr Vastus Medialis Median Frequency  pre-test-Ohr  *  0.135  t  p  3.81  0.005*  2.62  0.031*  5.90  <.001*  (df, 8) 0.060 (df, 8) 0.167  signficantly different from the pre-test.  144  References  1.  LindstrOm LH, Magnusson RI. Interpretation of myoelectric power spectra: a  model and its applications. Proceedings of the IEEE 1977;65(5):653-662.  2.  Zwarts MJ, Van Weerden TW, Haenen HTM. Relationship between average  muscle fibre conduction velocity and emg power spectra during isometric contraction, recovery and applied ischemia. European Journal of Applied Physiology 1987;56:212-216.  3.  Frascarelli M, Rocchi L, Feola I. EMG computerized analysis of localized fatigue in  duchenne muscular dystrophy. Muscle and Nerve 1988;11:757-761.  4.  Tarkka IM. Power spectrum of electromyography in arm and leg muscles during  isometric contractions and fatigue. Journal of Sports Medicine 1984;24: 189-194.  5.  Bigland-Ritchie B. EMG/force relations and fatigue of human voluntary  contractions. 1981:75-117. Exercise and Sport Science Reviews  6.  Delagi EF, Perotto A, lazetti J, Morrison D. Anatomic Guide for the  Electromyographer. Springfield, IL: C. Thomas, 1975  7.  Lieber RL. Skeletal Muscle Structure and Function.Baltimore: Williams and  Wilkins, 1992  8.  Glenberg AM. Learning From Data: An Introduction to Statistical Reasoning.New  York: Harcourt Brace Jovanovich, 1988  145  9.  Newham DJ, Mills KR, Quigley BM, Edwards RI-IT. Pain and fatigue after  concentric and eccentric contractions. Clinical Science 1983 ;64:55-62.  10.  Jones DA, Newham DJ, Torgan C. Mechanical influences on long-lasting human  muscle fatigue and delayed-onset pain. Journal of Physiology 1989;412:415-427.  11.  Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres during  contractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1921.  146  APPENDIX FOUR  Radio-isotope Investigation of Acute Inflammation  I Labelling procedure  Fifty millilitres (ml) of blood was taken by venipuncture from each of the subjects. In the lab the blood was separated into two tubes. One tube of 10 ml was centrifuged at 3000 revolutions/minute (rpm) for 20 minutes, while a surfactant was added to the rest of the blood and it was allowed to separate for 45 minutes. Platelet poor plasma (PPP) was removed from the 10 ml of blood, to be used in later stages. After 45 minutes leukocyte rich plasma (LRP) was removed from the sedimented red blood cells and centrifuged at 900 rpm for 10 minutes. After 10 minutes all of the plasma was removed from the leukocyte, or white blood cell (WBC), pellet. One ml of PPP was added to the pellet and the cells were re-suspended.  Next the Technetium-99m (Tc-99m) HMPAO labelling solution was prepared. The extract of Tc-99m was not more than four hours old. To a vial of HMPAO, 925 megabequerels (MBq) of Tc-99m was aseptically added. To make up a total volume of five ml, low 02 saline was added. This mixture was allowed to incubate for three minutes at room temperature. Following incubation, the next step was a liquid extraction quality control procedure to determine the percentage of HMPAO. Not more than 20% impurities or less than 80% HMPAO was found. From the mixture four ml (740 MBq) was drawn for the WBC labelling procedure.  The four ml Tc-99m HMPAO was added to the WBC, the cells were re-suspended, and incubated for 20 minutes. Following incubation, the cells were spun at 800 rpm for  147  ten minutes. Then the supernatant was removed, which contained the Tc-99m not labelled to the WBC. Following the removal of the supematant, the Tc-99m labelled WBC were re suspended with three ml of PPP.  The subject dose was drawn and a second quality control procedure was performed. In the dose calibrator, the activity of the supernatant and the cells was measured to obtain a labelling efficiency. Labelling efficiency is usually between 50 and 60%. The trypan blue exclusion test was done to determine the number of dead cells. Less than five dead cells should be seen in the field of view under the microscope. The dosages drawn for the subjects ranged from 235 454 MBq. -  II Analysis of the Presence of Tc-99m WBC Regions of Interest (ROl)  After the series of four scans was completed, computer analysis of regions of interest  (ROl) was used to determine the count/pixel of gamma radiation. Four areas of the quadriceps muscle were chosen as the ROT  -  the antero-distal aspect from the lateral view,  the anterior aspect from the lateral view, the medial aspect from the anterior view and the lateral aspect from the anterior view (Figure 4).  The analyses of the ROl were undertaken on three separate occasions by two scorers. The ROl were carefully drawn to avoid the femur and the femoral circulation. Figure 20 is an example of the mean count/pixel of the three analyses as well as the standard deviation (SD) for one subject for the antero-distal ROl. The figure illustrates the differences in the presence of Tc-99m WBC between the two legs, and the decay of the Tc 99m over time. For all subjects the exercise leg was the right leg and the non-exercise leg the left leg.  148  Figure 20. Subject 4: Antero-IDistal Region of Interest. Values are means ± SD. See text for explanation.  149  Figure 20  3o-  n I’ —-4-—  25  • -J  r.  2O  —  ci  Exercise Leg Non-exercise Leg —  I—  z C L_)  z  15  I —  I -  510152025 TIME (Hours)  150  The SD is a useful measure of variation within a given set of data. However, if there are large differences in the means within the data set a measure of relative variation allows comparisons to be made. The coefficient of variation (CV) expresses the SD as a percentage of the mean (SD/mean x 100) (1). Thus, the CV served as a measure of interobserver variability in this study. Table 16 is a summary of the mean count/pixel, as well as the SD, of each ROT at each test time for both legs for all of the subjects. Table 17 is a summary of the CV of each ROT at each test time for both legs for all of the subjects.  From Table 17 it can be seen that most of the variability occurred in the medial and lateral aspects as these were the ROT that were the most difficult to draw from an anatomical perspective. As well, the variability between observers increased at the later time periods as the scans were less well defined due to the decaying Tc-99m. The ROT with the least inter-observer variability was the anterior ROI.  Calculations  First the ROT of the non-exercise leg was subtracted from the ROl of the exercise leg to eliminate the background levels of Tc-99m. Table 18 summarizes the subtraction of the background for each ROT for each subject.  Next, the physical decay of the Tc-99m was corrected using a decay table. The calibration time, or the time at which the subject dose is drawn, is considered hour 0 and is the time at which 100% of the Tc-99m is present. From calibration time to the scan times of two, four, 20 and 24 hours were three, five, 21 and 25 hours respectively. The physical decay, or the fraction of the Tc-99m remaining, at those times were 0.708, 0.562, 0.089, and 0.056 respectively. The mean count/pixel of each ROT of the exercise leg, with the  151  TABLE 16 MEAN COUNT/PIXEL (± 1 SD) FOR EACH REGION OF INTEREST FOR EACH SUBJECT AT EACH TEST TIME ANTERO-DISTAL 2 Hours  Subject 1 2 3 4 5 6 7 8 9 10  Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left  15.56(0.3) 10.43(0.2) 14.73(0.7) 6.99(0.2) 54.00(0.3) 12.61(0.4) 28.36(4.4) 10.00(1.4) 24.40(2.1) 13.17(0.8) 13.55(1.0) 12.00(0.9) 22.23(1.7) 13.78(0.5) 29.59(2.9) 9.89(0.5) 67.55(4.2) 12.22(0.5) 123.33(2.5) 14.78(1.1)  4 Hours  20 Hours  24 Hours  11.36(0.3) 10.00(0.2) 11.47(1.3) 5.38(0. 1) 24.21(1.8) 7.20(0.4) 21.03(0.9) 9.51(0.3) 19.88(0.5) 11.39(0.3) 9.27(0.4) 7.60(0.5) 19.71(0.2) 11.10(0. 1) 25.33(1.9) 7.96(0.2) 63.98(2.9) 9.24(0.2) 89.21(3.4) 11.57(0.7)  2.63(0.5) 2.50(0.4) 3.19(0.2) 2.09(0.4) 7.95(1.0) 2.22(0.2) 4.86(0.3) 2.53(0.4) 4.97(0.3) 3.23(0.1) 2.99(0.4) 2.59(0.2) 5. 17(0.5) 3.05(0.2) 5.71(0. 1) 2.31(0. 1) 10.52(0.9) 2.49(0.3) 19.83(1.8) 2.90(0.3)  2.10(0.4) 1.72(0.2) 1.94(0. 1) 1.29(0. 1) 3.48(0.4) 1.55(0.1) 3.37(0.2) 1.93(0. 1) 3.19(0.2) 2.11(0.1) 2. 13(0.3) 1.75(0.4) 3.35(0.4) 2.42(0.5) 3.45(0. 1) 1.56(0.2) 2.55(0.1) 1.94(0.1) 9.01(5.9) 1.99(0. 1)  152  ANTERIOR Subjects  1 2 3 4 5 6 7 8 9 10  Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left  2 Hours  4 Hours  20 Hours  24 Hours  15.95(0.7) 13.97(0.8) 12.62(0.8) 9.60(0.7) 20.81(0.7) 13.49(1.1) 15.65(0.7) 12.54(0.6) 16.77(1.7) 14.60(1.2) 16.23(1.0) 13.09(1.2) 20.92(1.5) 16.49(1.1) 13.77(0.7) 10.32(0.6) 22.24(0.9) 14.07(1.2) 22.99(1.6) 17.38(1.7)  11.96(0.7) 10.74(0.4) 9.00(0.2) 8.00(0.7) 12.96(0.2) 9.87(0.6) 14.74(0.9) 9.43(0.8) 13.91(0.8) 12.57(0.6) 10.58(0.6) 9.71(0.3) 17.99(1.49) 13.12(1.1) 11.06(0.5) 9.41(0.6) 17.04(0.6) 11.26(0.8) 21.98(0.6) 13.80(0.6)  2.76(0.1) 2.70(0. 1) 2.63(0.3) 2.18(0.1) 4.11(0.1) 2.69(0. 1) 3.45(0.1) 2.59(0. 1) 3.66(0.1) 3.47(0. 1) 4. 10(0.4) 3.48(0.3) 6.39(0.4) 3.46(0.1) 2.85(0.1) 2.62(0.1) 4.53(0.2) 2.89(0.1) 6.53(0.5) 2.86(0.1)  2.15(0.1) 2.07(0. 1) 1.96(0.1) 1.60(0.3) 2.71(0.1) 2.09(0.1) 2.39(0.1) 1.84(0.1) 2.80(0.1) 1.84(0.6) 3.02(0. 1) 2.23(0.2) 4.36(0.1) 3.05(0.2) 1.95(0.1) 1.82(0.1) 3.05(0. 1) 2.28(1.1) 5.36(0.7) 2. 16(0.2)  153  MEDIAL  Subjects 1 2 3 4 5 6 7 8 9 10  Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left  2 Hours  4 Hours  20 Hours  24 Hours  20.70(2.8) 19.48(2.6) 11.11(2.3) 8.25(1.5) 33.31(3.0) 20.43(2.6) 18.32(1.6) 13.77(2.1) 21.86(2.9) 21.21(3.6) 17.61(3.2) 15.54(1.2) 19.94(2.7) 18.31(0.8) 13.49(0.4) 11.29(0.4) 26.13(8.3) 17.59(1.7) 32.62(3.0) 19.57(1.5)  13.73(1.0) 10.51(0.5) 10.35(1.1) 6.68(0.3) 18.19(5.6) 10.87(0.6) 20.64(1.4) 12.45(1.1) 12.85(2.9) 11.51(3.7) 10.98(2.2) 11.54(2.5) 14.91(3.6) 13.41(0.1) 11.92(1.0) 10.62(0.6) 22.58(7.4) 12.78(0.7) 26.03(8.5) 13.63(1.7)  3.21(0.4) 2.83(0.2) 3.73(0.1) 2.04(0.3) 6.42(0.5) 2.48(0.1) 3.91(0.3) 2.84(0.2) 3.84(0.4) 3.28(0.5) 4.05(1.3) 3.45(0.3) 5.80(2.1) 3.44(0.5) 2.73(0.3) 2.64(0.1) 6.24(3.2) 3.01(0.1) 5.86(0.7) 3.35(0.2)  2.54(0.1) 1.92(0.2) 2.13(0.2) 1.60(0.2) 3.96(0.2) 2.16(0.3) 2.84(0.5) 2.02(0.1) 3.07(0.7) 2.15(0.1) 2.87(0.7) 2.14(0.1) 3.53(1.4) 2.08(0.3) 1.85(0.2) 1.61(0.1) 3.77(1.1) 2.24(0.1) 3.71(0.7) 2.36(0.1)  LATERAL Subjects 1 2 3 4 5 6 7 8 9 10  Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left  2 Hours  4Hours  20 Hours  24 Hours  18.59(1.0) 19.25(2.1) 13.82(0.7) 11.42(0.4) 27.29(1.8) 20.63(1.3) 25.00(7.8) 16.69(1.3) 27.76(7.5) 2 1.43(2.5) 17.47(0.6) 16.15(0.7) 20.84(1.2) 19.66(2.0) 13.67(1.5) 12.47(0.7) 25.58(0.5) 19.87(1.5) 29.58(4.6) 18.35(5.5)  12.72(1.2) 13.50(1.0) 10.60(0.9) 9.18(0.5) 16.89(1.5) 11.60(1.8) 15.93(1.9) 12.25(1.2) 17.98(4.6) 14.28(2.0) 11.79(1.4) 12.52(1.0) 17.68(1.3) 13.89(2.3) 12.88(0.3) 10.55(1.1) 21.13(3.0) 13.59(0.4) 21.34(5.0) 14.74(4.2)  2.64(0.2) 2.49(0.5) 3.01(0.5) 2.30(0.1) 5.21(0.7) 2.71(0.4) 3.71(0.4) 2.64(0.3) 4.39(0.9) 3.80(0.1) 4.04(0.6) 4.10(0.4) 6.28(0.5) 3.43(0.4) 2.88(0. 1) 2.45(0.3) 4.95(0.5) 3.77(0.1) 4.66(0.5) 3.35(0.5)  2.29(0.2) 2.11(0.2) 1.96(0.3) 1.61(0.1) 2.98(0.3) 2.05(0.2) 2.64(0.2) 2.14(0.1) 3.09(0.7) 2.66(0.2) 2.55(0.5) 3.02(0.1) 3.78(0.4) 2.64(0.3) 2.00(0.2) 1.74(0.1) 3.13(0.2) 2.36(0.1) 3.21(0.8) 2.23(0.4)  4  3  2  24  4 20  5.93  24  1.67  2.90  6.17 15.81 2.07  6.78  3.16  4.76  4.47  3.69  2.43  1.54  3.36  4 20  15.51 14.00  12.58 9.01 14.37 6.45  2  24  20  4  10.37 3.17 7.43 5.55  4.59  7.29 8.75  4.81  3.70  3.72  4.89  1.16  8.48  4.78  4.78  3.72  6.08  8.15  5.10 18.75  11.41  6.27 19.14 5.15 7.75  6.34 2.22  2.86  4.65  3.62  5.85  Anterior R L 4.39 5.73  1.86  4.75 11.33  19.05 11.63  24  2  19.01 16.06  20  2.00  2.64  4  1.92  1.93  Antero-Distal R L  2  Hour  2  Subject  7.07  4.76  4.03  7.67 17.61  1.98  7.04  8.73 15.25 6.78 8.84  5.05 13.89  7.79  9.00 12.73 30.79 5.52  9.39 12.50  2.68 14.71  20.70 18.18 10.63 4.49  3.94 10.42  12.46  7.28  Medial R L 13.53 13.35  COEFFICIENT OF VARIATION (%) FOR THE REGIONS OF INTEREST  TABLE 17  9.48  20.00  7.40  6.30  9.80  7.79  9.76  10.78 11.36 7.58 4.67  11.93  31.20  10.07  8.88 15.52 13.44 14.76  6.60  15.31 6.21  5.07 3.50 8.49 5.45 16.61 4.35  8.73  7.58  9.43  Lateral R L 5.38 10.91  U,  9  8  7  6  5  9.67  20  9.80 7.90 1.75 2.90 6.22 4.53  8.56 3.92  2  4  20  24  2  4  20  24  24  1.02  4 0.90  3.63  7.72  5.15  12.05  2.16  4.09  12.82  4.33  2.51  5.06  6.56 11.94 20.66  7.65  14.08 22.86  24  2  13.38  6.58  20  7.50  2.37  4.31  6.27  24  3.10  4  6.04  20  2.63  7.38  2.52  4  6.07  2  8.61  2 0.58  4.77  8.22  3.28  4.42  3.52  4.05  5.13  3.51  4.52  5.07  2.29  6.26  8.34  7.17  3.31  9.76  5.67  6.16  4.39  3.47  7.10  8.53  5.49  3.82  6.38  5.81  6.56  2.89  8.38  6.67  8.97  8.62  3.09  9.17  1.79 32.61  2.73  5.75  10.14  7.72  4.19  4.37  4.67  8.70  29.18 4.46  51.28 3.32  32.77 5.48  31.76 9.66  10.81 4.67  10.99 3.79  8.39 5.65  2.97 3.54  0.75 36.21 14.53 39.66 14.42  24.14  13.54  24.39  32.10  20.03 21.66  18.17  22.80  10.42 15.24  22.57 32.15  13.27 16.97  7.99  4.33  7.52  5.61  5.75  6.39 4.24  10.10 2.65  14.20 2.94  1.95 7.54  10.00  3.47 12.24  2.33 10.43  10.97  10.58 11.36  5.76 10.17 7.35 14.40 7.96 11.66  19.61 13.57  14.85 15.34  11.87  3.43  22.65  25.58 14.01 20.50 1.32  27.02 11.67  ci, ci-’  10  24  65.56  9.08 10.34  20 5.03  6.05  3.80  4  7.43  2.03  2  13.06 9.26  7.66 3.50  2.73 4.35  6.96 9.78 7.66  18.87  11.95 4.24  5.97  32.65 12.47  9.20  24.92 17.94  10.73 14.93  23.43 28.49  15.55 29.97  I  157  TABLE 18 SUBTRACTION OF THE BACKGROUND (Exercise Leg Non- exercise Leg) -  Subjects  Hour  Antero-Distal  Anterior  Medial  Lateral  2 4 20 24  5.14 1.36 0.13 0.38  1.98 1.22 0.06 0.08  1.22 3.22 0.38 0.63  -0.66 -0.78 0.14 0.18  2  2 4 20 24  7.74 6.10 1.10 0.65  3.02 1.00 0.48 0.36  2.86 3.66 1.69 0.53  2.40 1.42 0.70 0.35  3  2 4 20 24  41.40 17.00 5.73 1.94  7.31 3.09 1.42 0.62  12.88 7.32 3.94 1.80  6.66 5.28 2.50 0.93  4  2 4 20 24  18.36 11.52 2.33 1.45  3.11 5.31 0.86 0.55  4.55 8.18 1.07 0.82  8.31 3.68 1.07 0.50  5  2 4 20 24  11.23 8.48 1.74 1.08  2.17 1.34 0.18 0.96  0.65 1.34 0.56 0.93  6.32 3.70 0.58 0.43  6  2 4 20 24  1.55 1.67 0.41 0.37  3.14 0.88 0.63 0.78  2.08 -0.56 0.60 0.35  1.32 -0.73 0.78 0.73  7  2 4 20 24  8.45 8.61 2.11 0.93  4.43 4.87 2.92 1.31  1.63 1.51 2.37 1.45  1.18 3.79 2.85 1.14  8  2 4 20 24  19.70 17.38 3.41 1.89  3.46 1.65 0.23 0.13  2.20 1.29 0.09 0.25  1.20 2.33 0.43 0.26  158  9  2 4 20 24  55.34 54.74 8.03 0.61  8.17 5.78 1.66 0.76  8.54 9.80 3.23 1.53  5.72 7.54 1.18 0.76  10  2 4 20 24  108.55 77.64 16.93 7.01  5.61 8.18 3.67 3.20  13.05 12.40 2.51 1.35  11.23 6.60 1.31 0.98  159  background subtracted, was divided by the fraction of Tc-99m remaining to arrive at the decay corrected values for each subject and each ROT (Figures 21 25). The figures for -  subjects 1 and 6 have a different Y scale to the others because they both had negative values. The Y scales for Figure 25 are also different because the presence of Tc-99m WBC in at least one of the ROT was much greater than for the other subjects.  Finally, because the dosages of Tc-99m WBC varied between subjects, the data were normalized to the peak response of each subject for each ROT. The non-normalized and normalized data are presented in Tables 19 and 20 respectively.  III Possible Mechanisms Contributing to the Responses of Two Subjects  The decay corrected differences for each ROT for two subjects subject 6 and -  subject 10- represent the extreme responses of this data set (Figures 23 and 25, Table 19). The presence of Tc-99m WBC in the antero-distal ROT for subject 10 ranged from 18 times greater at 24 hours post-exercise to 70 times greater at two hours post-exercise compared to the responses for subject 6. The magnitude of different responses in the presence of Tc 99m WBC were not as great in the other ROT but still they ranged from 1.68 times greater to 20 times greater numbers of Tc-99m WBC throughout the quadriceps muscle for subject 10 versus subject 6.  One of the major differences in the responses of the two subjects was that the presence of Tc-99m WBC in the quadriceps muscle was evident immediately at two hours post-exercise in subject 10 (Figure 25), while the responses were low in subject 6 until 20 hours post-exercise (Figure 23). The second major difference was that the presence of Tc 99m WBC in the antero-distal ROT of subject 10 was substantially greater than the other  160  The Presence of Technetium-99m White Blood Cells in the Four Regions of Interest for Each Subject. The background has been subtracted and the counts/pixel decaycorrected. The legends on each figure represent the four regions of interest. Figure 21. Subject 1. The scale on the y axis is the same as Subject 6, but different to the other subjects, because Subjects 1 and 6 both had negative values. Subject 2. Figure 22. Subject 3 and Subject 4. Figure 23. Subject 5 and Subject 6. The scale on the y axis for Subject 6 is the same as for Subject 1. Figure 24. Subject 7 and Subject 8. Figure 25. Subject 9 and Subject 10. The scale on the y axis is different for these two subjects from the other subjects because the presence of Tc-99m WBC in at least one ROT was greater than for the other subjects..  161 Figure 21 SUBJECT 1 70 Medial  —-•--  • -J Lu  51.5  Lateral  -  Anterior  a  Antero-Distal  -  -  z 33  —  Ci  z w 14.5  -  ._••_  -4  x  I  —  0  5  10  15  —  20  25  TIME (Hours)  SUBJECT 2 70  60  :  -.  t  • -J Lu ‘C  50  I—  z  D 0 (-3  Lateral Anterior  —• °---  t-  0  ±  ----Medial  -i  - -X- - Antero-Distal  -  40 —t-  + -  I  30  ±  i: III-  w  --1  20 — — .  10  _  *  t I  *  —  —  )E--X  -  -  0  0  5  10  15  TIME (Hours)  20  25  162  —--—  Figure 22  Lateral SUBJECT 3  -J  Ui  70  -  60  -  50  -  Anterior  - -X- -  Antero-Distal  \  — —  —  4  _...,.  —  —  4  -  — -  C) C)  — <>—  x  4  40  Medial  —  —  4.  —  —  —  30  Lii  20  -  10  -  0 0  5  10 15 TIME (Hours)  20  25  SUBJECT 4 70  I  ——  I  —  I  60  +  —-—MediaI •  -J Lii  50  E  >c  --- <>—-  - -  Lateral Anterior  - -  20  25  a I—  z  C) C)  z  40 30  Lii  20 10 0 0  5  10 15 TIME (Hours)  163 Figure 23  SUBJECT 5  70 60  -  50  -  —--—  • -J Ui  I—  z 0 Li  z  Lateral Anterior  —°---  a  -  -X-  40  Medial  -  Antero-Distal  30  4 UI  20  x  -  >6 - -  10  --  -  ew —  0 0  5  10 15 TIME (Hours)  20  25  SUBJECT 6 70 .-i  “Medial  ‘  I—  I -  -J UI  51.5  Lateral  S -  -  — °—  a  -  -X-  -  -4-  Anterior  -  Antero-Distal  I  z  0  33  ci  z  4  UI  z  14.5 -e-’..  --•--H  -4 0  5  10 15 TIME (Hours)  20  25  164 Figure 24 SUBJECT 7 70 60 T  Medial  —--—  -I  •  Lateral  -J  Anterior  ‘—-  ‘C  U  -  I—  40+  0 C-)  30  z z  -X-  -  Antero-Distal  -  ±  I—  -I  I  Lii  20 1-  10  4—  I  x —.  I—  -I  0 0  5  10  15 TIME (Hours)  20  25  SUBJECT 8 70 60  —  “—MediaI  ±  Lateral ----  -J Lii ‘C  50 —  U I 0 C-)  z  40  30 i:Fr  -x-----  -X-  -  Antero-Distal  .------  X  I  Lii  E  ± FF !  a---- Anterior  20  I  I—  10 L ‘—  0  -  0  — —.  —  5  10  15 TIME (Hours)  —  —  —(:—  20  —  25  _ _____  165 Figure 25  --•--  Medial  —  Lateral  SUBJECT 9  —X-  -  / -  80—  Anterior  “  I  100  -  -  -  Antero-Distal  -  x  >c J’J  I—  z  D L)  z  40 ___-.-—  I_u  20  — -  I  0-. 0  5  I  10 15 TIME (Hours)  I  -  20  25  Medial  —  Lateral Anterior  SUBJECT 1 0 200  -  -  -X-  -  Antero-Distal  --y’  -  1501  -  100— ci  z i_u  50 .,-—--—  -  o0  I5  10 15 TIME (Hours)  20  25  166  TABLE 19 TECHNETIUM-99M WHITE BLOOD CELLS FOR EACH REGION OF INTEREST AT EACH TEST TIME (Mean Count/Pixel) ANTERO-DISTAL  Subject  2 Hours  4 Hours  20 Hours  24 Hours  1 2 3 4 5 6 7 8 9 10  7.25 10.93 58.47 25.94 15.86 2.19 11.94 27.83 78.15 153.31  2.42 10.84 30.25 20.50 15.09 2.97 15.32 30.92 97.41 138.16  1.51 12.39 64.39 26.21 19.59 4.56 23.76 38.27 90.22 190.25  6.73 11.60 34.57 25.82 19.28 6.68 16.67 33.75 10.89 125.25  ANTERIOR Subjects  2 Hours  4 Hours  20 Hours  24 Hours  1 2 3 4 5 6 7 8 9 10  2.80 4.26 10.33 4.39 3.06 4.44 6.26 4.89 11.53 7.93  2.17 1.77 5.50 9.44 2.40 1.56 8.66 2.94 10.29 14.56  0.64 5.02 15.96 9.70 2.07 7.03 32.85 2.59 18.60 41.27  1.43 6.43 11.13 9.82 17.14 14.00 23.45 2.38 13.64 57.20  167  MEDIAL Subjects  2 Hours  4 Hours  20 Hours  24 Hours  1 2 3 4 5 6 7 8 9 10  1.72 4.04 18.20 6.43 0.92 2.91 2.29 3.11 12.07 18.43  5.74 6.52 13.02 14.56 2.39 -1.00 2.68 2.30 17.43 22.07  4.27 18.99 44.34 12.02 6.33 6.73 26.58 0.98 36.30 28.20  11.18 9.41 32.09 14.64 16.54 13.03 25.89 4.39 27.27 24.11  LATERAL Subjects  2 Hours  4Hours  20 Hours  24 Hours  1 2 3 4 5 6 7 8 9 10  -0.94 3.39 9.41 11.74 8.93 1.87 1.67 1.69 8.07 15.87  -1.39 2.54 9.40 6.55 6.58 -1.30 6.73 4.16 13.41 11.74  1.62 7.91 28.06 12.06 6.56 8.76 31.99 4.86 13.22 14.75  3.16 6.25 16.61 8.98 7.63 6.18 20.31 4.64 13.64 17.50  168  TABLE 20 NORMALIZED DATA FOR EACH REGION OF INTEREST  (%) ANTERO-DISTAL Subject  2 Hours  4 Hours  20 Hours  1 2 3 4  100.00 88.22 90.81 98.97 80.96 32.78  33.38 87.49 46.98 78.21 77.03 44.46 64.48 80.79 100.00 72.62  20.83 100.00 100.00 100.00 100.00 68.26 100.00 100.00 92.31 100.00  98.51 98.42 100.00 70.16 88.19 11.18 65.83  20 Hours  24 Hours  22.86 78.07 100.00 98.78 12.07 50.21 100.00 52.97 100.00 72.15  51.07 100.00 69.74 100.00 100.00 100.00 71.39 48.67 73.33 100.00  5 6 7 8 9 10  50.25 72.72 80.23 80.58  24 Hours 92.83 93.62  53.69  ANTERIOR Subjects  2 Hours  4 Hours  1 2 3 4 5 6 7 8 9 10  100.00 66.25 64.72 44.70 17.84 31.71 19.06 100.00 61.99 13.86  77.50 27.53 34.46 96.13 13.99 11.14 26.36 60.12 55.32 25.45  169  DIAL  Subjects 1 2 3 4 5 6 7 8 9 10  2 Hours  4 Hours  15.38 21.27 41.06 43.92 05.56 22.33 08.62 70.84 33.25 65.35  51.34 34.33 29.36 99.45 14.45 -7.67 10.08 52.39 48.02 78.26  20 Hours  24 Hours  38.19 100.00 100.00 82.10 38.27 51.65 100.00 22.32 100.00 100.00  100.00 49.55 72.37 100.00 100.00 100.00 97.40 100.00 75.12 85.50  LATERAL  Subjects  2 Hours  4Hours  20 Hours  24 Hours  1 2 3 4 5 6 7 8 9 10  -29.75 42.86 33.54 97.35 100.00 21.35 05.22 34.77 59.24 90.69  -43.99 32.11 33.50 54.31 73.68 -14.84 21.04 85.60 98.31 67.09  51.27 100.00 100.00 100.00 73.46 100.00 100.00 100.00 96.92 84.29  100.00 79.01 59.19 74.46 85.44 70.55 63.49 95.47 100.00 100.00  170  ROT for this subject (Figure 25), and was the greatest of any of the subjects (Table 19). The response also continued to be high over 24 hours. The presence of Tc-99m WBC in the antero-distal ROI of subject 6 was less than the other ROT for this subject, except at four hours post-exercise (Figure 23), and amongst the lowest responses of all of the subjects (Table 19).  As the dosages of Tc-99m WBC varied amongst the subjects, it might be suspected that subject 10 had a higher dosage than subject 6 but in fact the opposite was true. The dosage prepared for subject 10 was 331 MBq and for subject 6 it was 454 MBq Tc-99m WBC. In both cases the Tc-99m WBC were injected within one hour of calibration. The background levels and the physical decay of Tc-99m were accounted for in the data preparation for both subjects, as previously described.  Among the biological factors to be considered, the vascular response to the eccentric exercise protocol may have been different for the two subjects. Tt is not known if there was a difference between subjects in the blood flow to the muscle, which would normally be increased following exercise and also as a result of acute inflammation (2). Tt is also typical to have an increase in vascular permeability in response to acute inflammation (2) which may have caused swelling and subsequently decreased ROM. Subject 10 had an immediate decrease in ROM at two hours post-exercise which did not recover until 72 hours post-exercise, while ROM did not change for subject 6 until 20 hours post-exercise (Figure 26A).  Subject 10 had an immediately elevated response in the presence of Tc-99m WBC in the exercised muscle, particularly in the antero-distal ROI (Figure 25), while subject 6 had a gradual increase in the presence of Tc-99m WBC up to 20 and 24 hours post-exercise (Figure 23). The presence of greater numbers of WBC suggests that there was more  171  damage in the quadriceps muscle of subject 10 than subject 6. Evans and Cannon (3) have stated that the generation of an acute inflammatory response depends, at least to some extent, on the duration and on the intensity of exercise. Because the intensity was under the control of the subject, this may have been one of the factors contributing to the different responses for these two subjects.  Evans and Cannon (3) have stated that tissue damage or infection initiate a host of defense reactions that promotes clearance of the damaged tissue and sets the stage for repair. Complement is the name given to a complex series of plasma proteins which when triggered produces a rapid, highly amplified response to cell injury or invasion (4). The complement system can be activated by damaged host cells via the alternative pathway (3). Some of the end products of the complement cascade are C3a and C5a (4) which are activators of mast cells, neutrophils and monocytes (3). Following the vascular response, neutrophils are attracted to the area of tissue damage within minutes and they remain present in the tissue for up to 24 hours following the insult (3). Neutrophils are drawn to the site of injury by chemoattractants such as C5a (3).  Circulating monocytes derive into macrophages in the tissue (2). The lifespan of macrophages in the tissues varies from days to months (3). Neutrophils and macrophages both phagocytize, or clean up, the damaged tissue, however, in the process, neutrophils may also damage the host tissue (3). Macrophages, in addition to clearing the debris and the neutrophils, begin the process of repair and are important in the regeneration and stabilization of the tissue (3).  Figures 26 28 illustrate the responses of the functional measures of subject 6 and -  subject 10 compared to the group mean ± one SD. There was an immediate response in all of the functional measures for subject 10 and only ROM and fatigue of vastus lateralis (VL)  172  The Responses of Two Subjects Compared to the Group Mean ± One Standard Deviation. Figure 26. A. Range of Motion of the Right Knee. B. Eccentric Torque. Figure 27. A. Intensity of Soreness. B. Unpleasantness. Figure 28. A. Vastus Lateralis Median Frequency. B. Vastus Medialis Median Frequency.  C 1 0  0  x  m  C  Ni  0  -o -‘ CD  NJ 0  -  0  CT 0 0) 0  0 0  ECCENTRIC TORQUE (Nm)  N) 0 0  -  0 C 0  m  0)  cD r’J  -o CD  I  o  N)  \  -  ‘ f4  ‘ ‘  0  I  I I  ..-  ....,-_..T  (.*J  Ni C-n  -  I  0  -  ...  j-.-  .  W (‘1  RANGE OF MOTION (degrees)  I  (r >  -I,  0,  (D  u:•  U,  0  i’)  o  I  I,  0  -Jo_) r’  0  U, (I)  z rn  (I,  -  m  Z  D D .OO  -  o  H  F’)  0  r\j  -J  U,  I.  0  MEAN SCORE  U,  I I I  0 (11  (I,  0 C  H  co  0  Ni  Ni  (t  o -r  —  —  C --  — — — — — — —  f-i.  u’I  —  c-n  MEAN SCORE  I  --.  C  -  4  -  (‘1  U)  0  OD  r  0  ri  (t  Li  —  ro -  I  p  I  p (A)  b  0  I  SLOPE OF THE MEDIAN FREQUENCY  C,  C  p  (I)  0  .1,,  -o —I  OD  0  (tI  I  H  p  I  N  \  I  p  f_r._r-..-r--  p  I  p  I  T .4  -  I  p  S  I  SLOPE OF THE MEDIAN FREQUENCY  ,  I  ,  OD  -S (D  ‘-I  —4  I—  0,  176  had recovered by 72 hours post-exercise. Conversely, intensity of soreness, unpleasantness and ROM did not increase noticeably until 20 hours post-exercise for subject 6 and had recovered by 72 hours post-exercise. Eccentric torque had recovered to levels greater than the pre-test by four hours, and although it declined again at 24 hours post-exercise, the torque at 20, 48 and 72 hours post-exercise was also greater than the pre test. Slight fatigue was evident only at two hours post-exercise in both VL and VM for subject 6.  When the differences in the functional responses of the two subjects are considered in light of the substantial differences in the presence of Tc-99m WBC in the antero-distal ROT, the results suggest that the presence of Tc-99m WBC, and thus the extent of damage, in the distal segment of the quadriceps muscle may have been the most important factor contributing to the subjects’ different functional responses. The location of the antero distal ROT in this study is in the region of the musculo-tendinous junction of the quadriceps muscle. Stauber (5) has suggested that connective tissue damage is a component of exercise-induced muscle injury. However, there has been little study in this area and more direct evidence of connective tissue damage is needed.  Friden and colleagues (6) reported more pronounced Z line disorganization in human muscle three days after an eccentric exercise protocol than at one hour following the exercise, suggesting a progression of the tissue damage. Fritz and Stauber (7) reported degradation of proteoglycan, a component of extracellular matrix (ECM), in the area surrounding damaged myofibres of rats 24- 72 hours post-injury. The progression of damage in the muscle may be related to the activity of the WBC, especially neutrophils, at the site of injury. Neutrophils have little ability to differentiate between foreign and host cells (8). They also release an assortment of oxygen-dependent and oxygen-independent products that are capable of destroying normal cells and dissolving connective tissue (8).  177  Elastase and collagenase are two proteolytic enzymes that have the greatest potential for tissue damage (8). These enzymes attack the ECM, presumably to aid in migration of the neutrophils during the inflammatory response (9). As well, the neutrophil’s secretory and injurious potential at the site of injury may be amplified by priming agents from monocytes and macrophages or platelet activating factor (PAF) or tumor necrosis factor (TNF) (9). However, for healthy repair of tissue there must be a balance between tissue injury and protection of the tissue. In addition to antioxidants within the tissue, there are physical processes which are thought to be protective. Neutrophil migration into tissue ceases very early in the acute inflammation stage, chemoattractant activity may decrease, and neutrophils, both disintegrated and senescent, are actively removed by macrophages (9). It is not known how neutrophils and macrophages interact in damaged muscle but the progressive injury reported by others and the evidence of WBC within the muscle over 24 hours from this study suggest that this is an area of further study.  In summary, further research would benefit from collection of Tc-99m WBC data over a longer period of time, such as 48 hours. This would require a second dosage of Tc 99m WBC or use of another radio-isotope, such as Indium, which has a longer half life. Specific labelling of neutrophils and/or monocytes would allow specific description of the acute phase cellular response following eccentric exercise. More specific investigation of the musculo-tendinous junction following eccentric exercise may provide further information regarding the status of the connective tissue. And finally, if it were possible to keep the dosages of Tc-99m WBC constant then correlations with functional measures could be made which would also provide information about the importance of the location of the muscle injury.  178  References  1.  Dame! WW. Biostatistics: A Foundation for Analysis in Health Sciences. (5th ecL)  New York: John Wiley and Sons, 1991  2.  Kent TH, Hart MN. Injury, Inflammation and Repair.Norwalk, CT: Appleton and  Lange, 1993  3.  Evans WJ, Cannon JG. The metabolic effects of exercise-induced muscle  damage.Baltimore: Williams and Wilkins, 1991:99- 125. Exercise and Sports Science Reviews  4.  Roitt I. Essential Immunology. (7 ed.) London: Blackwell Scientific Publications,  1991  5.  Stauber WT, Clarkson PM, Fritz VK, Evans WJ. Extracellular matrix disruption  and pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.  6.  Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentric  exercise in man. International Journal of Sports Medicine 1983 ;4: 170-176.  7.  Fritz VK, Stauber WT. Characterization of muscles injured by forced lengthening.  II. Proteoglycans. Medicine and Science in Sports and Exercise 1988;20(4):354-361.  8.  Weiss SJ. Tissue destruction by neutrophils. New England Journal of Medicine  1989320(6) :365-375.  179  9.  Haslett C, Savill JS, Meagher L. The neutrophil. Current Opinion in Immunology  1989;2: 10-18.  180  APPENDIX FIVE Individual Subject Data Chapter One -  =  missing data  Subject 1 Age  42  Gender  F  Weight (Kg)  68. 1  Leg Tested  R  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  8.32  14.68  9.32  8.73  DDS Unpleasant.  -10  0.77  2.53  2.82  0.61  VAS  *  *  *  *  *  Ecc. Torque (N.m)  124  77  84  85  95  CPK (lUlL)  55  397  340  455  3034  OHP (umol/L)  39  33  63  51  62  Cr(umol/L)  3034  3507  4175  3969  3918  OHPICr  12.8  9.4  15.0  12.8  15.8  48  96  168  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  DDS Soreness  -10  3.86  14.21  9.49  -6.91  DDS Unpleasant.  -10  -5.71  0.82  0.52  -8.32  VAS  *  *  *  *  *  Eec. Torque (N.m)  125  109  98  113  113  CPK (lUlL)  48  240  136  458  408  01-P (umol/L)  93  86  88  51  90  Cr (umollL)  4466  6435  6218  5061  6460  01-P/Cr  20.8  13.4  14.1  10.1  13.9  181  Subject 2 Age  42  Gender  F  Weight (Kg)  71.7  Leg Tested  R  Physical Activity (Hr/wk)  0  Exercise Duration  Shorter (180 reps) 96  168  1.11  -7.23  -9.00  -9.33  -1.15  -8.46  -9.00  *  *  *  *  *  Ecc. Torque (N.m)  108  92  101  118  126  CPK (lUlL)  77  103  76  60  94  OHP (umollL)  93  77  82  94  160  Cr (umollL)  6224  6608  5828  6795  7424  OHPICr  14.9  11.7  14.1  13.8  21.6  96  168  Test Session (Hour)  pre-test  24  DDS Soreness  -10  -2.24  DDS Unpleasant.  -10  VAS  48  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  DDS Soreness  -10  10.05  5.93  2.71  -10  DDS Unpleasant.  -10  0.32  4.86  -2.36  -10  VAS  0.0  4.3  5.9  1.4  0.0  Eec. Torque(N.m)  114  68  65  82  110  CPK (lUlL)  35  99  86  95  97  Ol-IP (umollL)  137  53  59  68  48  Cr(umol/L)  4381  5410  5035  7199  6613  OHP/Cr  31.3  9.8  11.7  9.5  7.3  182  Subject 3 Age  20  Gender  F  Weight (Kg)  55.8  Leg Tested  L  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -3.79  15.61  16.82  14.39  -10  DDS Unpleasant.  -10  6.82  6.52  -6.67  -10  VAS  0.0  7.8  5.0  2.9  0.0  Ecc. Torque (N.m)  138  84  99  113  124  CPK (lUlL)  58  216  148  250  *  01-P (umol/L)  139  524  302  240  255  Cr(umol/L)  7707  17668  12713  13586  10978  01-P/Cr  18.0  29.7  23.8  17.7  23.2  96  168  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  DDS Soreness  -1.05  14.99  14.23  6.12  -10  DDS Unpleasant.  -10  0.77  3.73  -3.76  -10  VAS  *  *  *  *  *  Eec. Torque (N.m)  144  80  96  124  125  CPK (lUlL)  60  311  131  123  59  01-P (umol/L)  244  174  367  225  338  Cr(umol/L)  12519  9705  9609  8120  10519  01-P/Cr  19.5  17.9  38.2  27.7  32.1  183  Subject 4 Age  24  Gender  F  Weight (Kg)  49.9  Leg Tested  R  Physical Activity (Hr/wk)  1  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -9  -7.14  -6.52  -4.67  -8.20  DDS Unpleasant.  -9  -6.99  -8.09  -7.99  -8.94  VAS  0.0  0.6  1.3  0.4  0.4  Ecc. Torque (N.m)  43  91  70  66  57  CPK (lUlL)  44  52  49  57  41  01-P (umol/L)  349  344  191  204  245  Cr(umollL)  11059  13759  12425  9457  13737  OHPICr  31.6  25.0  15.4  21.6  17.8  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -9.18  10.5  11.44  14.44  -8.32  DDSUnpleasant.  -10  10.17  10.36  12.52  -10  VAS  0.2  8.6  7.3  5.5  0.0  Ecc. Torque (N.m)  58  39  87  70  76  CPK (lU/L)  67  132  78  44  32  01-P (umol/L)  357  250  190  353  148  Cr(umol/L)  11292  11162  8263  12336  7060  01-P/Cr  31.6  22.4  22.9  28.6  20.9  184  Subject 5 Age  48  Gender  F  Weight (Kg)  56.8  R Leg Tested Physical Activity (Hrlwk)  0  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  DDS Soreness  -5.71  13.83  11.64  7.27  -7.49  DDS Unpleasant.  -9.71  -0.29  -1.89  -7.65  -9.14  0.2  2.3  1.6  2.3  1.4  VAS  96  168  Ecc. Torque (N.m)  108  80  94  97  113  CPK (lUlL)  72  176  135  106  106  OHP (umollL)  *  46  40  44  47  Cr(umol/L)  *  4987  4157  3259  5163  OHP/Cr  *  9.2  9.6  13.5  9.1  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -4.17  10.91  14.21  -4.02  -9.18  DDS Unpleasant.  -9.14  -4.49  -3.99  -8.71  -10  0.0  2.5  4.6  0.9  VAS Eec. Torque (N.m)  66  55  CPK (lU/L)  83  158  62 113  OHP (umol/L)  65  94  Cr (umol/L)  8445  OI-IP/Cr  7.7  0.9  63  79 123  43  81 48  46  5327  4948  4160  4838  17.65  8.7  11.54  9.51  185  Subject 6 Age  27  Gender  M  Weight (Kg)  70.4  Leg Tested  R  Physical Activity (Hr/wk)  4  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  DDS Soreness DDS Unpleasant.  -8.77  9.86  11.91  12.53  8.21  -9.38  7.86  10.58  3.47  -5.67  0.2  5.5  7.3  6.8  2.3  VAS  168  Eec. Torque (N.m) CPK (lU/L)  177  119  85  118  122  154  324  281  340  726  OHP (umol/L)  174  362  355  341  225  Cr(umol/L)  14531  20175  20965  20187  19835  OHP/Cr  19.7  17.9  16.9  16.9  11.3  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -7.52  14.38  13.97  *  -9.09 -10  DDS Unpleasant.  -10  6.61  9.36  *  VAS  0.0  5.0  6.6  *  0.0 168  Ecc. Torque (N.m)  165  104  100  *  CPK (lUlL)  121  208  180  *  1257  Ol-IP (umol/L)  100  190  247  *  40  Cr(umollL)  12608  16327  20064  *  10332  OHPICr  7.9  11.6  12.3  *  39  186  Subject 7 Age  21  Gender  F  Weight (Kg)  59  Leg Tested  R  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  DDS Soreness  -10  10.70  9.61  DDS Unpleasant.  -10  -3.02  -0.46  VAS  0.0  5.0  5.0  Ecc. Torque (N.m)  146  88  76  93  *  CPK (lUlL)  *  140  *  48  *  OI-IP(umol/L)  431  425  220  114  *  Cr(umol/L)  9618  17981  6889  7432  *  OHPICr  44.8  23.6  31.9  15.3  *  48  96  168  Exercise Duration  48  96  168  -0.23  *  -10  *  1.1  *  Longer (300 reps)  Test Session (Hour)  pre-test  24  DDS Soreness  -10  6.71  0.99  -10  -10  DDS Unpleasant.  -10  -5.29  -9.56  -10  -10  VAS  0.0  2.3  0.1  0.0  0.0  Ecc.Torque(N.m)  77  112  118  83  123  CPK (lU/L)  34  131  *  53  88  OHP(umol/L)  159  469  230  364  356  Cr(umollL)  12560  19393  11126  14080  17152  OHPICr  12.6  24.2  20.6  25.8  20.7  187  Subject 8 Age  38  Gender  F  Weight (Kg)  48.6  Leg Tested  R  Physical Activity (Hr/wk)  1  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  DDS Soreness  -10  DDS Unpleasant. VAS  24  48  96  168  1.53  13.32  -10  -10  -10  -12.42  -5.52  -10  -10  0.0  1.8  0.9  0.0  0.0  Ecc.Torque(N.m)  118  84  87  117  126  CPK(IU/L)  *  *  *  *  *  OHP(umollL)  167  75  106  151  79  Cr (umol/L)  5681  6993  6410  5481  7476  OHPICr  29.0  10.7  16.5  27.5  10.5  48  96  168  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  DDS Soreness  -10  6.42  DDS Unpleasant.  -10  -9.47  -10  VAS  0.0  6.4  1.4  Eec. Torque (N.m)  74  75  104  101  106  CPK (lUlL)  61  152  157  129  148  OHP(umollL)  97  83  57  115  53  Cr (umollL)  8926  8536  4641  5806  5806  OHPICr  10.8  9.7  12.2  19.8  9.1  24  -0.73  4.14  -7.91  -9.43  -9.09  1.1  0.0  188  Subject9 Age  23  Gender  F  Weight (Kg)  62.2  Leg Tested  R  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps)  Test Session (Hour) DDS Soreness DDS Unpleasant. VAS  pre-test  24  48  96  168  1.44  12.30  14.61  -4.79  -9.67  -9.15  -2.00  1.11  -10  -10  1.7  3.6  4.1  0.3  0.2  Eec. Torque (N.m)  108  60  95  88  101  CPK (lUlL)  52  147  80  60  53  OHP(umollL)  67  112  296  179  113  Cr(umol/L)  2723  5397  7877  8104  4410  OHPICr  24.6  20.8  37.6  22.1  25.6  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -0.88  13.11  13.03  -10  -10  DDS Unpleasant.  -6.09  -0.85  2.38  -8.42  -10  1.2  6.4  7.9  0.0  0.0  VAS  Eec. Torque (N.m) CPK (lUlL)  104  75  50  97  107  63  192  196  1245  610  OI-IP(umolIL)  164  145  126  214  301  Cr(umol/L)  6453  7940  5748  9917  11781  OHPICr  25.4  18.3  21.9  21.5  25.5  189  Subject 10 Age  19  Gender  F  Weight (Kg)  53.6  Leg Tested  R  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  DDS Soreness  -8.26  DDS Unpleasant. VAS  24  48  96  168  5.20  9.56  -0.47  -9.76  -8.70  -1.21  6.77  -3.35  -10  1.6  5.7  5.9  2.7  0.1  Eec. Torque(N.m)  57  99  81  100  119  CPK (lUlL)  152  503  345  145  113  OHP(umol/L)  147  227  370  110  286  Cr (umol/L)  7225  9549  10679  6268  12575  OHP/Cr  20.3  23.7  34.6  17.5  22.7  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -6.88  9.88  10.00  7.61  -4.86  DDS Unpleasant.  -9.65  6.39  10.24  8.62  -8.08  0.7  7.7  9.6  5.9  0.9  VAS Eec. Torque (N.m)  110  102  *  81  105  CPK (lU/L)  81  493  248  318  480  OHP(umol/L)  91  72  165  200  101  Cr (umol/L)  5337  6047  6338  8005  2993  OI-IP/Cr  17.0  11.9  26.0  24.9  33.7  190  Subject 11 Age  20  Gender  M  Weight (Kg)  77.2  Leg Tested  R  Physical Activity (Hr/wk)  0  Exercise Duration  Shorter (180 reps) 24  48  96  168  Test Session (Hour)  pre-test  DDS Soreness  3.59  5.15  14.49  0.05  -9.47  DDS Unpleasant.  -10  -5.23  0.77  -8.26  -10  VAS  0.0  4.8  5.5  2.7  0.0  Ecc.Torque(N.m)  180  144  156  208  221  CPK (lUlL)  79  296  189  204  100  OHP (umollL)  343  255  520  257  147  Cr(umol/L)  17809  27571  23148  18532  18906  OHPICr  19.3  9.3  22.5  13.9  7.8  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -7.06  13.64  11.38  15.24  -6.86  DDS Unpleasant.  -10  -3.15  13.26  4.08  -7.70  VAS  0.5  5.6  8.4  5.8  1.4  Ecc.Torque(N.m)  187  150  52  135  202  CPK (lU/L)  88  396  2057  2545  369  OHP(umol/L)  307  313  171  361  153  Cr(umollL)  18188  27384  19449  17862  16516  OHPICr  16.9  11.4  8.8  20.2  9.3  191  Subject 12 Age  28  Gender  F  Weight (Kg)  65.8  Leg Tested  R  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  DDS Soreness  -9.21  14.06  14.30  8.62  DDS Unpleasant.  -10  2.17  -1.74  -10  VAS  0.1  1.52 9.6  6.4  4.1  0.9  Eec. Torque (N.m)  84  69  83  121  136  CPK (lUlL)  91  791  424  785  553  OHP (umol/L)  56  130  57  45  297  Cr(umollL)  5996  10315  7232  6769  9369  OHPICr  9.3  12.6  7.9  6.7  31.7  Exercise Duration  96  168 -7.47  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  13.29  10.74  14.24  -4.21  DDS Unpleasant.  -10  2.99  10.88  -1.70  -10  VAS  0.0  8.4  9.8  5.0  2.8  Eec. Torque (N.m)  128  78  70  102  104  CPK (lUlL)  125  2043  4545  18070  2839  Ol-IP (umollL)  *  34  98  99  69  Cr(umol/L)  *  5170  8969  7581  7785  OHP/Cr  *  6.6  10.9  13.1  8.9  192  Subject 13 Age  43  Gender  M  Weight (Kg)  66  R Leg Tested Physical Activity (Hrlwk)  0  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  *  12.3  9.97  13.68  6.96  DDS Unpleasant.  -9.23  8.06  4.33  2.20  -3.49  0.9  8.4  *  59  1.1  VAS Ecc. Torque(N.m)  153  57  83  77  115  CPK (lU/L)  72  650  490  926  928  OI-IP(umol/L)  68  84  80  81  100  Cr (umol/L)  5932  8307  4932  6243  12590  OI-IPICr  11.5  10.1  16.2  12.9  7.9  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -9.52  13.29  10.00  12.03  -9.47  DDS Unpleasant.  -10  1.83  1.58  1.00  VAS  0.2  8.4  5.9  Eec. Torque (N.m)  134  73  CPK (lU/L)  38  OI-IP(umol/L)  -10 0.2  99  2.5 99  354  224  181  140  199  149  88  167  111  Cr(umol/L)  8137  10799  5138  8384  5769  OI-IP/Cr  24.5  13.8  17.1  19.9  19.2  155  193  Subject 14 Age  52  Gender  F  Weight (Kg)  58.1  Leg Tested  L  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  96  168  DDS Soreness  -10  3.91  4.49  -8.30  -10  DDS Unpleasant.  -10  -8.50  -4.55  -10  -10  VAS  0.0  1.8  3.6  0.2  0.0  Ecc.Torque(N.m)  132  78  94  103  108  CPK (lU/L)  42  82  53  32  163  OHP(umol/L)  162  179  91  128  57  Cr (umol/L)  8925  8832  8223  6639  5887  OHP/Cr  18.2  20.3  11.1  19.3  9.7  48  96  168  Exercise Duration  24  48  Longer (300 reps)  Test Session (Hour)  pre-test  24  DDS Soreness  -10  1.62  3.82  -10  -10  DDS Unpleasant.  -10  -9.71  -2.15  -10  -10  VAS  0.0  1.8  3.4  0.0  0.0  Eec. Torque (N.m) CPK (lUlL)  132  91  100  105  92  27  195  136  68  38  OHP(umolIL)  96  185  104  82  Cr(umollL)  5001  193 11929  9331  9098  5813  OHPICr  19.2  16.2  19.8  11.4  14.1  194  Subject 15 Age  20  Gender  F  Weight (Kg)  93.1  Leg Tested  L  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  11.89  11.92  13.27  -10  DDS Unpleasant.  -10  10.96  11.42  1.52  -10  VAS  0.0  8.9  9.1  2.7  0.0  Ecc. Torque (N.m)  125  69  62  115  162  CPK (lUlL)  46  *  102  178  159  Ol-IP (umol/L)  47  195  88  159  209  Cr(umollL)  5088  10484  5790  9186  10892  OHPICr  9.24  18.6  15.2  17.3  19.2  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  12.56  11.39  -0.55  -10  DDS Unpleasant.  -10  1.55  6.5  -8.85  -10  VAS  0.0  6.4  6.4  0.9  0.0  Eec. Torque (N.m)  101  111  116  160  165  CPK (lUlL)  60  192  112  68  51  OHP(umollL)  155  255  143  171  132  Cr(umol/L)  9475  17781  11685  8131  8589  OHPICr  16.4  14.3  12.2  21.0  15.4  195  Subject 16 Age  34  Gender  M  Weight (Kg)  74.9  Leg Tested  R  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  13.82  13.40  14.05  -10  DDS Unpleasant.  -10  1.26  11.68  3.70  -10  VAS  0.0  4.2  5.9  2.3  0.0  Ecc. Torque (N.m)  236  176  159  223  266  CPK (lUlL)  137  380  188  79  86  OHP (umol/L)  36  73  63  53  20  Cr(umollL)  4688  12132  14462  4959  3134  OHPICr  7.7  6.0  4.4  10.7  6.4  96  168  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  DDS Soreness  -10  8.55  14.23  5.41  -10  DDS Unpleasant.  -10  0.53  8.15  -8.56  -10  VAS  0.0  3.6  7.1  2.1  0.0  Ecc. Torque (N.m)  209  167  200  256  231  CPK (lUlL) Ol-IP (umollL)  106  755  356  104  81  82  72  74  79  66  Cr(umol/L)  10128  7442  6722  9926  7041  OI-IPICr  8.1  9.7  11.0  7.9  9.4  196  Subject 17 Age  23  Gender  F  Weight (Kg)  59  Leg Tested  R  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  DDS Soreness  -10  9.33  12.12  4.27  -4.24  DDS Unpleasant.  -10  -0.55  3.18  -3.70  -7.86  VAS  0.0  4.1  5.0  3.2  Ecc.Torque(N.m)  128  72  74  87  100  CPK (lUlL)  20  120  80  90  120  OI-IP (umol/L)  60  198  62  167  162  Cr (umollL)  3536  7319  3855  8129  9246  OHPICr  16.9  27.0  16.1  20.5  17.5  Exercise Duration  24  48  96  168  0.0  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  10.08  11.62  9.26  -7.52  DDS Unpleasant.  -10  0.86  3.08  0.05  -10  VAS  0.0  3.7  5.5  1.8  0.0  Eec. Torque (N.m)  128  87  85  88  108  CPK (lUlL)  20  162  104  131  61 168  Ol-IP (umol/L)  185  96  170  *  Cr (umollL)  7561  6699  9462  7454  7261  17.9  *  23.1  OHPICr  24.5  14.3  197  Subject 18 Age  40  Gender  F  Weight (Kg)  50  Leg Tested  R  Physical Activity (Hr/wk)  2  Exercise Duration  Shorter (180 reps) 96  168  7.39  -4.67  -10  2.76  -1.26  -7.50  -10  5.0  3.2  0.7  0.0  48  Test Session (Hour)  pre-test  24  DDS Soreness  -10  12.26  DDS Unpleasant.  -10  VAS  0.0  Ecc. Torque (N.m)  68  37  45  60  63  CPK (lU/L)  20  34  20  20  20  OHP (umol/L)  68  21  38  54  62  Cr(umol/L)  6836  4167  4170  6956  7830  OI-IP/Cr  9.9  5.0  9.1  7.8  7.9  Longer (300 reps)  Exercise Duration Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -8.99  -0.30  -9.33  -7.30  -7.08  DDS Unpleasant.  -10  -6.88  0.00  -8.47  -8.33  VAS  0.0  5.0  4.0  0.7  0.0  Ecc. Torque (N.m)  35  41  47  49  49  CPK (lU/L)  29  48  23  20  20  OHP (umol/L)  128  69  60  50  77  Cr(umol/L)  5313  5184  6335  4103  7976  OI-IP/Cr  24.1  13.3  9.5  12.2  9.6  198  Subject 19 Age  24  Gender  F  Weight (Kg)  54.5  Leg Tested  R  Physical Activity (Hr/wk)  3  Exercise Duration  Shorter (180 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  8.12  7.92  4.70  -10  DDS Unpleasant.  -9.09  2.35  2.52  -2.06  -10  0.1  5.0  5.3  1.8  0.1  VAS Ecc. Torque (N.m)  80  37  34  83  103  CPK (lUlL)  43  165  82  60  68  OHP(umolIL)  99  121  72  167  50  Cr(umol/L)  4679  8416  5949  7351  2835  OHPICr  21.2  14.4  12.1  22.7  17.6  Exercise Duration  Longer (300 reps)  Test Session (Hour)  pre-test  24  48  96  168  DDS Soreness  -10  8.12  7.92  4.70  -10  DDS Unpleasant.  -10  3.12  3.53  -6.64  -8.49  VAS  0.0  7.3  8.4  1.2  Eec. Torque (N.m)  82  54  66  81  78  CPK (lUlL)  40  108  58  62  47  Ol-IP (umol/L)  *  *  37  102  147  Cr (umollL)  *  5549  3038  *  *  OHPICr  *  *  12.2  *  *  0.0  =  missing data  0.30  -7.97 -10 140  -0.280 -0.252 pretest 117  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM(slope)  Test Session (Hour)  Ecc. Torque (N.m) 59  0  -0.236  -0.259  140  75  2  2  pretest  Test Session (Hour) DDS Soreness -6.61  Longer (300 reps)  R  Leg Tested  Exercise Duration  54.0  Weight (Kg) 4.5  F  Gender  Physical Activity (Hr/wk)  33  Age  Subject 1  -0.269  -0.274  140  -5.15  7.68  4  79  4  -0.106  -0.133  135  -0.73  13.50  20  58  20  Individual Tc-99m WBC data is included in Appendix Four  *  -  Individual Subject Data Chapter Two  -0.095  -0.110  135  -1.71  14.91  24  60  24  -0.09  -0.118  130  14.62 -0.67  48  72  48  -0.145  -0.121  135  -0.40  10.29  72  85  72  I—  0 0  1.52 -5.27 135 -0.403 -0.414 pretest 138  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM (slope)  Test Session (Hour)  Ecc.Torque(N.m) 101  0  -0.497  -0.586  135  -3.80  149  2  2  pretest  Test Session (Hour) 6.89  Longer (300 reps)  R  Leg Tested  Exercise Duration  70  Weight (Kg) 5  F  Gender  Physical Activity (Hr/wk)  21  Age  Subject 2  -0.404  -0.407  135  -3.49  5.58  4  141  4  -0.719  -0.437  135  3.76  5.26  20  133  20  -0.856  -0.370  130  -1.36  11.59  24  107  24  -0.299  -0.353  130  -1.68  10.46  48  153  48  -0.723  -0.399  135  -0.82  11.23  72  136  72  -10 -10 135 -0.328 -0.253 pretest 99  DDSSoreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM(slope)  Test Session (Hour)  Eec. Torque (N.m) 57  0  -0.338  -0.329  135  -4.32  108  2  2  pretest  Test Session (Hour) 12.09  Longer (300 reps)  R  Leg Tested  Exercise Duration  72.7  Weight (Kg) 5  F  Gender  Physical Activity (Hr/wk)  28  Age  Subject3  -0.354  -0.372  130  -3.42  9.97  4  99  4  -0.279  -0.300  125  6.41  14.94  20  73  20  -0.228  -0.269  125  0.96  11.33  24  70  24  -0.210  -0.260  125  2.96  16.11  48  73  48  -0.196  -0.294  130  -0.99  5.20  72  83  72  N)  -9.52 -10 140 -.337 -.262  pretest 88  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM (slope)  Test Session (Hour)  Ecc. Torque (N.m)  .232  65  0  -  -.249  135  -8.97  60  2  2  pretest  Test Session (Hour) 4.41  Longer (300 reps)  R  Leg Tested  Exercise Duration  47.7  Weight (Kg) 1  F  Gender  Physical Activity (Hr/wk)  23  Age  Subject 4  -.190  -.238  135  -7.21  6.42  4  61  4  -.142  -.188  140  9.65 -5.79  20  53  20  -.073  -.112  135  -1.86  11.73  24  56  24  -.068  -.155  140  -8.27  6.03  48  71  48  -  .226  -.213  140  -10  -6.79  72  77  72  I\.)  r’.)  -8.74 -10 145 -.100 -.278 pretest 86  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM (slope)  Test Session (Hour)  Ecc. Torque (N.m) 59  0  -.230  -.227  140  -1.50  76  2  2  pretest  Test Session (Hour) 14.86  Longer (300 reps)  R  Leg Tested  Exercise Duration  52.3  Weight (Kg) 4.5  F  Gender  Physical Activity (Hr/wk)  29  Age  Subject 5  -.640  -.232  140  -1.33  12.14  4  86  4  -.29 1  -.197  140  1.00  13.80  20  65  20  -.295  -.195  135  2.17  14.50  24  65  24  -.224 -.449  145  -3.08  10.79  48  74  48  -.299  -.210  -2.79 145  11.88  72  84  72  cJ3  -9.47 -10 140 -.106 -.139 pretest 42  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM (slope)  Test Session (Hour)  Ecc. Torque (N.m) 29  0  -.144  -.110  140  -6.79  41  2  2  pretest  Test Session (Hour) -7.61  Longer (300 reps)  R  Leg Tested  Exercise Duration  60.5  Weight (Kg) 4  F  Gender  Physical Activity (Hr/wk)  26  Age  Subject 6  -.024  -.058  140  -6.26  -8.79  4  56  4  .028  -.026  135  4.77  -2.64  20  48  20  .002  -.050  130  10.64  10.36  24  38  24  -.035  -.043  130  12.82  12.33  48  67  48  -.067  -.085  140  -9.80  -9.18  72  54  72  -0.42 130 -0.218  -9.14 -10 140 -0.140 -0.139 pretest 132  DDS Soreness  DDS Unpleasant. ROM (degrees) VL(slope)  VM(slope)  Test Session (Hour)  Eec. Torque (N.m) 63  0  -0.232  116  2  2  pretest  Test Session (Hour) 10.65  Longer (300 reps)  R  Leg Tested  Exercise Duration  64.0  Weight (Kg) 1.5  F  Gender  Physical Activity (Hr/wk)  20  Age  Subject 7  -0.194  -0.234  135  0.92  10.38  4  118  4  -0.150  -0.189  135  1.96  7.52  20  121  20  -0.135  -0.169  135  3.30  10.30  24  126  24  -0.195  -0.231  135  0.47  5.52  48  171  48  -0.184  -0.226  140  -1.29 -7.52  72  167  72  I\.)  cD ci,  -0.91 -4.05 135 -0.143 -0.024 pretest 133  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM(slope)  Test Session (Hour)  Eec. Torque (N.m) 104  0  -0.13 1  -0.159  135  -2.92  80  2  2  pretest  Test Session (Hour) 1.50  Longer (300 reps)  R  Leg Tested  Exercise Duration  65.0  Weight (Kg) 3  F  Gender  Physical Activity (Hr/wk)  30  Age  Subject 8  0.059  -0.118  135  0.92  2.30  4  109  4  -0.045  -0.097  135  1.96  3.29  20  81  20  0.032  -0.044  135  3.47 3.30  24  24 108  0.020  -0.093  135  0.47  0.91  48  121  48  -0.041  -0.109  135  -7.52  2.56  72  115  72  N)  -9.67 -10 150 -0.3 14 -0.277 pretest 112  DDS Soreness  DDS Unpleasant.  ROM (degrees)  VL(slope)  VM (slope)  Test Session (Hour)  Eec. Torque (N.m) 69  0  -0.076  -0.384  150  -3.47  97  2  2  pretest  Test Session (Hour) 13.35  Longer (300 reps)  R  Leg Tested  Exercise Duration  62.0  Weight (Kg) 1.5  F  Gender  Physical Activity (Hr/wk)  29  Age  Subject 9  -0.065  -0.420  150  0.53  14.32  4  89  4  -0.198  -0.334  140  -0.26  14.62  20  64  20  -0.193  -0.421  145  2.14  15.08  24  53  24  -0.208  -0.235  140  4.62  14.88  48  75  48  -0.260  -0.368  145  3.17  15.73  72  84  72  r’)  F  54.5  Gender  Weight (Kg)  -0.413  -8.39 135 -0.345 -0.143  DDS Unpleasant.  ROM (degrees) VL(slope)  Test Session (Hour) Ecc. Torque (N.m) 0 53  pretest 122  -0.183  58  2  128  5.17  Test Session (Hour) DDS Soreness  VM (slope)  7.92  pretest  Exercise Duration  2.47  Longer (300 reps) 2  3  R Physical Activity (Hr/wk)  Leg Tested  27  Age  Subject 10  -0.188  -0.430  130  4.44  8.88  4  56  4  -0.255  -0.3 19  125  5.94  9.47  20  61  20  -0.172  -0.3 14  125  7.20  7.86  24  56  24  -0.278  -0.324  128  6.24  10.94  48  60  48  -0.266  -0.247  133  7.02  9.77  72  57  72  C  

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