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Effect of isometric training with short or long rest periods on Achilles tendon morphology : an ultrasound… Alketbi, Thuraya 2016

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       Effect of Isometric Training with Short or Long Rest Periods on Achilles Tendon Morphology: An Ultrasound Tissue Characterization Study    by THURAYA ALKETBI  B.Sc., University of Sharjah, 2004      A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE  in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Rehabilitation Science)        THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2016    © Thuraya Alketbi, 2016 Abstract Achilles tendon (AT) is the strongest tendon in the body. One of the major causes of AT pathology, chronic tendinopathy, is frequently due to excessive loading. Despite the risk of overuse injury, exercise training with appropriate mechanical loading parameters has been found to trigger an adaptation response in the AT. However, the influence of some loading parameters, such as the duration of rest periods between exercise repetitions, remains unknown. Therefore, the aim of this study was to examine the effect of 10s rest insertion during isometric resistance training (Long rest training; LRT) on the morphological properties of human AT, compared with same training intensity and volume but with 3s rest insertion (short rest training; SRT). I hypothesized that ATs exposed to LRT would demonstrate increased tendon collagen organization and tendon cross-sectional area (CSA) compared with baseline, but that SRT would not demonstrate any change in either variable. To test my hypothesis, we recruited healthy adults, and randomly allocated one leg to LRT and the other to SRT. The exercise protocol consisted of 5 sets of 10 isometric calf presses at 90% of maximum voluntary contraction, three times a week over a 12-weeks training period. Tendon collagen organization (proportion of type I echoes) and CSA were measured by ultrasound tissue characterization before and after the 12-weeks period. The primary outcome was the proportion of type I echoes. In contrast to our hypothesis, neither LRT nor SRT induced an improvement in collagen organization or tendon CSA. However, tendons exposed to SRT demonstrated a reduction in the proportion of type I echoes following exercise from 0.66 (95% CI= 0.60 to 0.70) to 0.55 (95% CI=0.49 to 0.60), (p < 0.05), whereas tendons that were exposed to LRT didn’t demonstrate any reduction in the proportion of type I echoes from baseline (0.60, 95% CI= 0.56 to 0.65) to post-intervention (0.62, 95% CI= 0.57 to 0.67), (p = 0.58). Neither type  ii of exercise had a measurable effect on the AT CSA. Further research is needed to examine the biological changes and mechanical effects that influence tendon morphological change associated with different exercise parameters.    iii Preface The University of British Columbia’s Clinical Research Ethics Board granted ethics approval for this research on February 13, 2015. The ethics approval certificate number for the current study is (H15-00043). To date, the research included in this thesis has not been published in full.     iv Table of Contents Abstract ................................................................................................................................. i Preface ................................................................................................................................. iii List of Tables ...................................................................................................................... vii List of Figures .................................................................................................................... viii List of Abbreviations and Acronyms .................................................................................... ix Acknowledgements ............................................................................................................... x Dedication ............................................................................................................................ xi Chapter 1. Introduction ......................................................................................................... 1 1.1 Overview ............................................................................................................... 1 1.2 Achilles Tendon Gross Anatomy ........................................................................... 3 1.3 Achilles Tendon Structure...................................................................................... 3 1.4 Achilles Tendon Composition ................................................................................ 5 1.5 Achilles Tendon Mechanical Properties ................................................................. 6 1.6 Tendon Physiological Response to Mechanical Loading ........................................ 7 1.7 Effect of Exercise and Training on Tendon Mechanical Properties ......................... 9 1.7.1 Tendon Cross-Sectional Area (CSA). ................................................................. 9 1.7.2 Regional Difference in Cross-Sectional Area (CSA). ....................................... 11 1.7.3  Collagen I .......................................................................................................... 11 1.7.4 Stiffness .............................................................................................................. 13 1.7.5 Young’s Modulus ............................................................................................... 14 1.8 Effect of Rest-Insertion Between Cyclic Load...................................................... 14 1.9 Effect of Immobilization on the Tendon ............................................................... 15 1.10 Effect of Aging on the Tendon ............................................................................. 16 1.11 Effect of Sex Difference on Tendon Properties .................................................... 17 1.12 Ultrasound Tissue Characterization ...................................................................... 18 1.13 Objectives ............................................................................................................ 22 1.13.1 Aim of the Study. ............................................................................................. 23 1.13.2 Hypotheses. ..................................................................................................... 23  v 1.14 Significance of the Research ................................................................................ 23 Chapter 2. Methods ............................................................................................................. 24 2.1 Participants .......................................................................................................... 24 2.2 Randomization ..................................................................................................... 25 2.3 Blinding ............................................................................................................... 26 2.4 Experimental Set-up ............................................................................................ 26 2.4.1 Pre-/Post-Training ............................................................................................ 26 2.4.2 Training Intervention ....................................................................................... 27 2.5 Ultrasound Tissue Characterization (UTC) .......................................................... 30 2.5.1 Measurement of the CSA. ................................................................................ 30 2.5.2 Measurement of Collagen Organization. .......................................................... 31 2.6 Validity and Reliability ........................................................................................ 32 2.6.1 Ultrasound Tissue Characterization (UTC)....................................................... 32 2.6.2 Isokinetic Dynamometer. ................................................................................. 32 2.6.3 IPAQ-Short Form. ........................................................................................... 32 2.7 Data Analysis ...................................................................................................... 33 2.7.1 Sample Size ..................................................................................................... 33 2.7.2 Statistical Analysis. .......................................................................................... 33 Chapter 3. Results ............................................................................................................... 35 3.1 Participant Allocation and Drop-out ..................................................................... 35 3.2 Compliance.......................................................................................................... 37 3.3 Participants Physical Activity .............................................................................. 37 3.4 Type I Echoes ...................................................................................................... 38 3.5 Cross-Sectional Area ........................................................................................... 40 3.6 Muscle Strength ................................................................................................... 42 3.7 Effect of Long Rest Exercise on Echo-Pattern ...................................................... 43 3.8 Effect of Short Rest Exercise on Echo Pattern ...................................................... 44 Chapter 4. Discussion .......................................................................................................... 45 4.1 Type I Echoes Alteration in SRT ......................................................................... 45  vi 4.2 Alteration in Type II and III Echoes? ................................................................... 46 4.3 Alteration in Echo Types as An Adaptive Response? ........................................... 47 4.4 Long Rest Insertion and Changes in Echo Type I ................................................. 48 4.5 CSA and Mechanical Load .................................................................................. 48 4.6 Did LRT Provide Sufficient Stimulus to Cause AT Adaptation? .......................... 49 4.7 Missing Data ....................................................................................................... 49 4.8 Limitations .......................................................................................................... 50 4.9 Future Directions ................................................................................................. 51 Chapter 5. Conclusion ......................................................................................................... 52 References .......................................................................................................................... 53 Appendix A: International Physical Activity Questionnaire Form ....................................... 67 Appendix B: Participant Characteristics and Activities ........................................................ 70 Appendix C: IPAQ Analysis Result..................................................................................... 72 Appendix D: Echo-Types Measurements............................................................................. 73 Appendix E: CSA Measurements ........................................................................................ 78 Appendix F: Muscle Strength Measurements ...................................................................... 91 Appendix G: Training Load Measurements ......................................................................... 94     vii List of Tables Table 1: Participant Characteristics; cm=centimetres, kg=kilograms. Values are Mean ± SD. .......................................................................................................................... 36 Table 2: Participant Activity Level Based on IPAQ Classifications. .................................... 38 Table 3: CSA Measurements Before and After 12 Weeks of Resistance Training; Presented in cm as LS Means (95% CI). .................................................................. 40       viii List of Figures Figure 1: Schematic Drawing of the Multi-Unit Hierarchical Structure of the Tendon (Wang, 2006). ............................................................................................................ 4 Figure 2: Tendon Stress-Strain Curve (Wang, 2006). ............................................................ 7 Figure 3:  Mechanisms of Tendon CSA Increase with Loading  (Gumucio et al., 2014) . ....... 9 Figure 4:  Experimental Set-Up ........................................................................................... 29 Figure 5: Biodex Setting. ..................................................................................................... 30 Figure 6: CSA of Achilles Tendon. ..................................................................................... 31 Figure 7: Flow Diagram of Participant Allocations, Follow-Up, and Analyses. ................... 36 Figure 9: Effect of Resistance Exercise on the Cross-Sectional Area of the Achilles Tendon. ................................................................................................................... 41 Figure 10: Muscle Strength (torque) Percentage of Change from Baseline According to Type of Training. ..................................................................................................... 42 Figure 11: Effect of Long Rest Training on Ultrasound Tissue Characterization (Proportion of Echo-Types). .................................................................................... 43 Figure 12: Effect of Short Rest Exercise on Tissue Characterization (Echo-Types). ............ 44        ix List of Abbreviations and Acronyms AD Agnatha DeSa AGE  Advanced Glycation End Products AP Anterior Posterior Diameter AS Alex Scott AT Achilles Tendon CI Confident Interval CSA Cross-Sectional Area CTGF  Connective Tissue Growth Factor ECM Extracellular Matrix IPAQ International Physical Activity Questionnaire LMM Linear Mixed Model LOX Lysyl Oxidase LRT Long (Rest insertion, 10s) Rest Training LS mean Least Square means mCSA Mean Cross-Sectional Area MET Metabolic Equivalent of Task MRI Magnetic Resonance Image MVC  Maximum Voluntary Contraction OCs Oral Contraceptives PGs Proteoglycans PICP Serum carboxyterminal propeptide of type I procollagen PINP Procollagen I N-Terminal Peptide RT Rest training SD Standard Deviation SDFT Superficial Digital Flexor Tendon SE  Standard Error SRT Short (Rest insertion, 3s) Rest Training TA Thuraya Alketbi TGF-β1  Transforming Growth Factor β1 US Ultrasound  x Acknowledgements I would like to thank Dr. Alex Scott for the amazing opportunity of being a graduate student in his lab as well as for all his advice and guidance throughout my time as a graduate student. His constant support, understanding, and generosity has kept me going through the rough patches, and his tremendous teaching ability has been invaluable. Special thanks to Dr. Charlie Waugh for the training and mentorship during this study.  I would like thank my other members of the supervisory committee, Dr. Maureen Ashe and Dr. Jordan Guenette for their support and advice. I am also very grateful to the entire the staff at the Centre for Hip Health and Mobility for providing necessary lab training and helping with anything I needed. Finally, I would like to thank the most important people in my life, my mother, sisters and friends. Without their love and support, I would not be where or who I am today.     xi  Dedication To my family, and my son Ahmad. 1 Chapter 1. Introduction 1.1 Overview Voluntary human movement arises from the forces generated by skeletal muscle contraction, which are transmitted via tendon to bone. Tendons are able to withstand high levels of tensile and compressive force, and can sustain forces up to 17-times body weight  (Maffulli et al., 2005). One of the strongest tendons in the body is the Achilles tendon (AT), which is regularly exposed to very high levels of tensile force, up to 12.5-times body weight during running (Arya & Kulig, 2010). Although the mechanical properties of the AT allow it to withstand tremendous forces, it has a high rate of injury in sporting and non-sporting populations, compared to other tendons in the body  (Maganaris & Paul, 2002; Wren, Yerby, Beaupré, & Carter, 2001).  In vivo investigations of the mechanical properties of human tendons have shown that they can adapt to changes in the physiologic loading pattern (e.g., low or high intensity, and disuse or immobilization) (Kubo, Kanehisa, & Fukunaga, 2002; Keitaro Kubo, Kanehisa, & Fukunaga, 2002). These authors and others have shown that tendons are responsive to loading and they can alter their structure and function based on the loads placed upon them. The exerted external load can cause an increase in the internal tensions at the cellular level; this internal tension is hypothesized to regulate gene expression in tendon cells and, if increased beyond a “calibration set point,” will cause the cells to engage adaptive physiologic responses (Lavagnino & Arnoczky, 2005). According to this theory, mechanical loads above the set point would facilitate anabolic processes, and the absence of mechanical loads, or loads that are below this point, would elicit catabolic processes in tendon tissue (Lavagnino et al., 2006). Loads can thereby influence the mechanics and morphology of the tendon by stimulating the cells to alter the tendon cross-sectional area (CSA) and the degree of stiffness  2 or by changing the material properties of tendon by changing the degree of intermolecular cross-linking. In the context of increased physiologic loading, these adaptations would act to improve the tendon’s resistance to strain, thereby returning the strain level experienced by cells to their set point (Heinemeier & Kjaer, 2011). The sum total of all tendon mechanical or/and morphological changes depends on the characteristics of the applied loads (magnitude, rate, duration, and frequency) delivered by exercise mode and dose. Many studies have investigated the effect of different loading conditions (magnitude, duration of single loading cycle, repetitions, sets, intervention duration and frequency) and exercise conditions (i.e. type of muscle contraction; isometric, eccentric, concentric). However, a recent systematic review by Bohm et.al (2015) concluded that interventions applying a loading magnitude higher than 70% of MVC are most effective in inducing tendon adaptation, with only a minor effect of type of contraction (Bohm, Mersmann, & Arampatzis, 2015).  However, very few studies have addressed the effects of controlled loading conditions on tendon adaptation such as high vs low magnitude/frequency and duration of rest periods during cyclic exercise (Bohm et al., 2015). Arampatzis et al., (2007, 2010) demonstrated that loads delivered at a low frequency which resulted in high magnitude strains were most able to trigger adaptive responses in the AT, as indicated by changes in mechanical, morphological, and material properties. Moreover, a recent study by Huisman et al., (2014) found that inserting a 10s rest period between cyclic loads (delivered in an optimized high strain / low frequency regimen) stimulates tenocyte collagen production at the gene and protein level more than the same loading regimen without rest periods. This finding may indicate a potential benefit of rest-inserted loading in the tendon adaptive response if applied in vivo. The investigation of potential implications of these changes in tendon function and performance could prevent injury and assist in the design of suitable exercises for rehabilitation.  3 1.2 Achilles Tendon Gross Anatomy The AT is the strongest and thickest tendon in the human body with an average length of approximately 15 cm (Maffulli, Renström, & Leadbetter, 2005). It begins near the middle of the calf, and is formed by the conjunction of the gastrocnemius and soleus muscles. The shape of the AT varies from the proximal end to the distal end as it approaches its calcaneal attachment. It is broad and flat at the gastrocnemius-soleus junction and as it travels distally it becomes ovoid in cross section, before returning to a relatively flattened shape at 4 cm proximal to its insertion. As the tendon descends, and depending on the fusion of the gastrocnemius and soleus muscles, the fibers of the AT rotate internally in a spiral manner up to 90 degrees. This is believed to enhance the tendon’s elastic recoil properties for the storage and release of energy during locomotion (Doral et al., 2010; Nunley & Nickisch, 2009). The gastrocnemius lies superficial to the soleus and consists of two heads, medial and lateral, that arise from the posterior aspect of the medial and lateral femoral condyles. The two heads also have an attachment to the posterior capsule of the knee. The soleus lies deep and anterior to the gastrocnemius, arising from the posterior aspect of the tibia and fibula and originating as a tendinous arch between the tibia and fibula. The AT is not covered by a true synovial sheath like other tendons, but by a paratenon that consists of a discontinuous synovial membrane (more present posteriorly) and loose areolar tissue (Calleja & Connell, 2010). 1.3 Achilles Tendon Structure The AT is mainly composed of a dense pack of Type I collagen fibers with a multi-unit hierarchical structure, from the molecular to the macroscopic level, arranged in parallel to its long axis. The collagen fibrils in the tendon have a zigzag or wavy crimped pattern that can be seen using polarizing microscopy (Kastelic, Galeski, & Baer, 1978). The multi-unit hierarchical structure of the tendon is composed of collagen fibrils, which are the smallest  4 tendon units; these fibrils are strong and stiff (Koob & Summers, 2002). A group of collagen fibrils forms a collagen fiber, which is considered the basic functional unit of the tendon. A group of collagen fibers then forms a primary fiber bundle (sub-fascicle), and a group of primary fiber bundles forms a secondary fiber bundle (fascicle). Thereafter, a group of secondary fascicles forms a tertiary bundle and the tertiary bundles make up the tendon (Kannus, 2000). The fascicles are surrounded by endotenon and the tendon is enveloped with epitenon, which is surrounded by the paratenon. A thin layer of fluid separates the epitenon from the paratenon to reduce the friction during tendon motion (Doral et al., 2010). The endotenon, a thin layer of connective tissue, contains blood vessels, lymphatics, and nerves, while the epitenon is a sheath of fine and loose connective tissue, containing vascular tissue, lymphatics, and a nerve supply to the tendon (Wang, 2006) (Figure1). Collagen in the matrix is cross-linked and the parallel organization of the collagen fibrils with the intermolecular cross-linking provides the tendon with its high tensile strength.   Figure 1: Schematic Drawing of the Multi-Unit Hierarchical Structure of the Tendon (Wang, 2006).  5 1.4 Achilles Tendon Composition The main components of the tendon are tenocytes (specialized fibroblasts), which produce the ECM. The ECM is composed of collagen fibers, elastic fibers, the ground substance, and trace components (Kannus, 2000).  Tenocytes comprise 90-95% of the cellular elements of the tendon. They are elongated cells that are located among the collagen fibers, which interact with each other and with adjacent collagen fibers, and which produce collagen fibers as well as release enzymes such as lysyl oxidase, contributing to the formation of collagen cross-links (Wang, 2006). Tenocytes are mechano-responsive, because of their ability to sense mechanical loading and respond by modulating the ECM through the alteration, formation or degradation of matrix proteins in a process called mechanotransduction (Kirkendall & Garrett, 1997; Chiquet et al., 2003). Collagen and elastin are the major components of the ECM, forming 70% and 2% of the dry weight of tendon, respectively (Doral et al., 2010). Collagen type I is the major fibrillar component of the tendon, and collagen type III is found mainly in the endotenon and epitenon (James et al., 2008). The ground substance consists of proteoglycans (PGs), glycosaminoglycans (GAGs), plasma proteins, water, and other small molecules that give the tendon its viscoelastic properties. Proteoglycans are considered secondary components of the tendon and comprise 1-20% of the dry weight of the tendon (Kirkendall & Garrett, 1997). They are a heterogeneous group of molecules that has at least one chain of GAGs that attached to the protein core. They have very important functions including water retention, modifying the activity of growth factors, and mediating cell-matrix interactions (Riley, 2008) . Examples of proteoglycans are aggrecan and decorin. Aggrecan is hydrophilic, and the water binding capacity of the ground substance can improve the elasticity of the tendon as well as its ability to resist shear and compressive forces (Riley, 2008). The decorin is a leucine-rich  6 proteoglycan located on the surface of the middle portion of collagen fibrils and it facilitates fibrillar slippage during mechanical deformation  (Iozzo & Schaefer, 2015). The anorganic components, such as calcium and copper, form less than 0.2% of the tendon dry mass and they have a role in growth, development, and metabolism in musculoskeletal structures (Kannus, 2000). 1.5 Achilles Tendon Mechanical Properties The unique structure and composition of the tendon contributes to its biomechanical behavior, including its high mechanical strength and viscoelasticity. This allows the tendon to carry and transmit forces from muscle to bone, permitting joint motion and stabilization. The tendon also acts as a buffer to protect and limit muscle injury by absorbing external forces (Sharma & Maffulli, 2006). Advances in the application of real-time ultrasonography have made it possible to identify and characterize the mechanical properties of human tendon in vivo (Maganaris & Paul, 2002). Relevant mechanical properties of tendons include tendon strain, stress, Young’s modulus, and stiffness. These parameters are measured in vivo during voluntary isometric contraction (Narici, Maffulli, & Maganaris, 2008). Strain is the relative change in tendon length, and it is expressed as the ratio or percentage of tendon length change compared to its resting length (Heinemeier & Kjaer, 2011). Stress is measured as the ratio of the tendon force to tendon CSA (Heinemeier & Kjaer, 2011). Young’s modulus is determined from the slope of the tendon stress-strain curve, and it mainly depends on the material properties of the tendon (Heinemeier & Kjaer, 2011). Tendon stiffness is measured as the slope of the force-deformation curve, and it is thus influenced by the tendon CSA and length (Heinemeier & Kjaer, 2011). A stress-strain curve demonstrates the mechanical behavior of the tendon, which reflects the tendon’s basic material characteristics. A typical stress-strain curve has three regions. A toe region represents the stretching out of the crimp pattern of collagen fiber  7 bundles as seen by polarized microscopy when the tendon is strained by as much as 2%. The second region, called the linear region, occurs where the collagen fibers lose their crimp pattern and the collagen fibers begin to stretch (Jozsa, 1997). The third region occurs at the end of the linear region (the yield point), where microscopic tearing of collagen fibers begins to occur. A macroscopic failure occurs when the tendon is strained at a value that exceeds its ultimate tensile stress (Wang, 2006) (Figure 2). The strain values that define the crimp, linear, and failure zones may vary from tendon to tendon, and depending on the method in which strains are visualized and calculated (e.g., in vitro vs in vivo).   Figure 2: Tendon Stress-Strain Curve (Wang, 2006).  1.6 Tendon Physiological Response to Mechanical Loading Tendons need to maintain a tensile strength higher than the maximum force generated by the combination of muscle and ground-reaction forces in order to withstand the maximum load placed on them (Koob & Summers, 2002). As mentioned above, tendons and other  8 connective tissues are characterized by their ability to sense mechanical forces and generate adaptive responses in order to maintain tensile strength which is adequate but not excessive (Chiquet, Renedo, Huber, & Flück, 2003). In other words, tendons are mechano-responsive and can alter their structure and function in response to the loads that are placed on them by up- or down-regulating anabolic and catabolic processes. Appropriate mechanical loads at physiological levels are beneficial to tendons since they stimulate ongoing anabolic processes (e.g., collagen synthesis), whereas mechanical loads that are beyond physiological levels are by definition harmful. It has been theorized that they lead to damage of the load-bearing ECM – this damage is accompanied by a subsequent catabolic process in tendons (e.g., matrix degradation) due to local regions of tenocytes becoming unloaded as a result of lack of force transmission in the region of the microrupture  (Schechtman & Bader, 2002; Wang, 2006). Therefore, mechanical loading within physiological limits is an important tool that promotes tissue homeostasis and remodeling, whereas excessive loading can lead to degeneration (Andarawis‐Puri, Flatow, & Soslowsky, 2015).  Tenocytes are connected with their surrounding ECM through a class of load-bearing cell-surface receptors known as integrins (Kjær, 2004). Forces are transmitted from the tendon ECM to the cytoskeleton via the integrins. The transmission of force across the integrins leads to the activation of cell signaling pathways which culminate in the production and release of growth factors. The mechanism by which collagen I synthesis increases with mechanical loading has been linked to several autocrine growth factors including transforming growth factor β1 (TGF-β1), connective tissue growth factor (CTGF) and interleuken-6 (IL-6) (Heinemeier & Kjaer, 2011). TGF-β1 has been measured with a microdialysis technique in human AT before and after treadmill running exercises, and it was found to be elevated after exercise, when compared to controls, in association with increased formation of type I collagen (Heinemeier & Langberg, 2003). Scleraxis is a transcriptional  9 regulator that is relatively specific to tendon cells, which is induced by TGF-beta signaling, and regulates the transcription of the gene for type I collagen. In a recent study, Scott et al., (2011) used a bioartificial tendon over a three-week period and reported a greater increase of tenocyte gene expression including scleraxis mRNA after applying cyclic (as opposed to static) mechanical loading. Despite the above studies, the mechanisms underpinning tendon adaptation are only just beginning to be elucidated. Indeed, recent studies suggest that tendon growth/hypertrophy in response to exercise may involve an increased production of ECM by resident cells, and the recruitment of perivascular stem cells into the tendon, where they differentiate into tenocytes – the characteristics of this process and the cells responsible have not yet been completely described  (Gumucio et al., 2014) (Figure 3).  Figure 3:  Mechanisms of Tendon CSA Increase with Loading  (Gumucio et al., 2014) .  Increase of total CSA of AT tendon after increased tendon loading. The growth of plantaris tendon (following surgical ablation of synergists) occurred throughout the formation of a neotendon matrix between the original tendon and epitenon, containing cells that were proliferative and Scleraxis positive.  1.7 Effect of Exercise and Training on Tendon Mechanical Properties 1.7.1 Tendon Cross-Sectional Area (CSA). A larger tendon CSA can help reduce the stress (force/area) of applied external forces, which would thereby reduce the magnitude of strain on the tendon and help protect the  10 tendon against overload injuries (Kongsgaard et al., 2007). Many studies have investigated the effect of training on tendon CSA, and some authors found that CSA changes only occur after high magnitude resistance training (Arampatzis et al., 2007)  or with long-term habitual training (Rosager et al., 2002; Magnusson & Kjaer, 2003) . In terms of habitual training, Magnusson and Kjaer (2003) conducted cross-sectional study to compare the CSA of tendons of runners who performed distance running at 80 km per week for the last 5 years to those of non-runners who perform a physical activity not more than twice a week. The authors found that the AT CSA was 36% greater in runners than in non-runners, in the most distal part of the tendon. Similar findings were observed in a study looking at habitual runners (chronic exposure to repetitive loading for more than 5 years) and the AT CSA was 22% larger than age-matched non-runners  (Rosager et al., 2002).  Arampatzis et al., (2007) conducted a controlled modulation of cyclic strain magnitude and found that high strain versus low strain isometric exercises caused a region-specific increase in the CSA of the AT. The increases in CSA were found to occur at specific locations (60% and 70% of the AT length) after 14 weeks of isometric strength training. A subsequent experiment was performed by the same group with similar exercise protocol on their previously described high strain isometric exercise but with different loading  frequencies, i.e., comparing high versus low frequencies (0.17 Hz vs. 0.5 Hz). The authors found that only the combination of high magnitude and low frequencies produced superior CSA adaptation compared to high frequency (Arampatzis, Peper, Bierbaum, & Albracht, 2010) . These studies indicate that the magnitude and frequency of loading may influence the extent of tendon adaptation. However, studies that have investigated the effect of shorter-term exercise programs have not found any changes in tendon CSA after 2-3 months or more of increased physical loading. For example, the CSA of the human AT did not change after 12 weeks and 14 weeks of plyometric training that consisted of series of jumping protocol and  11 weight training (Fouré, Nordez, & Cornu, 2010; Kubo et al., 2007). Similar results were found for moderately active subjects who performed planter flexion resistance training 3-times a week for 8 weeks (Urlando & Hawkins, 2007). Taken together, the evidence indicates that high magnitude loading (muscle contraction intensity) and longer-term interventions or habitual training (over years) are beneficial for eliciting changes in tendon CSA (Bohm et al., 2015). 1.7.2 Regional Difference in Cross-Sectional Area (CSA). The CSA of the AT varies along its length; the proximal segment is smaller than the distal segment. Consequently, the applied stress during exercise or other mechanical loading would be expected to vary along the length of the tendon, although this regional variation has not been well studied. Increased tendon CSA has been demonstrated with specific exercise regimens (see above) and evidence also indicates that this adaptation occurs in a region-specific manner. Magnusson and Kajer (2003) found significant variation in AT CSA along its length for runners who ran 80 km for the past 5 years, compared to controls. The most distal part of the AT was 36% larger than the controls, whereas the proximal regions were not significantly different. Arampatzis et al., (2007) also found that the distal portion of the AT was larger than the proximal part in the leg that exercised at a high cyclic strain (90% of maximum voluntary contraction; MVC), compared to the leg that exercised at low cyclic strain (55% of MVC) after 14 weeks of isometric strength training. 1.7.3  Collagen I Evidence for increased collagen production with loading has been found using different measurement approaches, such as microdialysis, tendon biopsies, and the analysis of mRNA expression (Kjaer et al., 2009; Heinemeier & Kjaer, 2011). Some authors found that a single bout of acute exercise (1 hr of strenuous kicking exercise i.e repetitive knee extension) and a long-term exercise (12 wks of strengthening exercises) increased collagen synthesis  12 (Miller et al., 2005; Langberg, Rosendal, & Kjær, 2001). Using microdialysis, Langberg et al., (1999) measured peritendinous collagen levels after acute exercise of AT (36 km of running) and found that a reduction in collagen I synthesis marker levels (serum carboxyterminal propeptide of type I procollagen, PICP) occurred immediately after exercise, but this was followed by a 3-fold increase after 72 hrs. The same group of authors measured collagen synthesis in the AT after 4 weeks and 12 weeks of strength training to determine whether prolonged training would increase the formation of collagen I (Langberg et al., 2001). The results confirmed their previous work showing that collagen synthesis increased after exercise and remained elevated throughout the entire training program. The increase in collagen turnover and the net collagen synthesis is hypothesized to underpin improvements in the mechanical properties of the AT to reduce the tendon stress (Fratzl & SpringerLink ebooks - Engineering, 2008). Moreover, as an adaptation effect on the tendon, collagen fibrils increases in diameter and this gives a greater ability to withstand the tensional forces. This is thought to be due in part to the higher number of intra-fibrillar covalent cross-links (Michna & Hartmann, 1989). An increase in collagen content and fibril diameter was also found in the flexor digitorum longus in mice after 10 weeks of running (Michna & Hartmann, 1989). As mentioned previously, the mechanical strength of the tendon is provided by collagen, a load-bearing molecule, and mechanical loading increases the induction of collagen synthesis. Nevertheless, collagen cross-linking is also a central factor giving the tendon its strength, stiffness, and toughness (Fessel, Gerber, & Snedeker, 2012). This cross-linking is in part regulated by lysyl oxidase (LOX), which forms stable intermolecular (covalent) collagen cross-linking during maturation and contributes to the tendon’s substantial tensile strength and integrity (Bailey, 2001). These intermolecular cross-links prevent the individual collagen molecules from sliding apart when a mechanical load is applied (Avery & Bailey, 2005). Studies have shown an increase in tensile strength and  13 stiffness occurs with acute and long-term exercise, possibly because of the increased collagen turnover and resulting increase in intermolecular cross-linking. In vitro study of human tendon, blocking LOX in early fibrillogenesis led to spontaneous rupture of tendon-like construct, whereas the un-blocked LOX in the control group showed an improvement in structure and strength seen by time  (Herchenhan, Uhlenbrock, & Eliasson, 2015) .  Also, evidence of increased expression of LOX is associated with an increase in tendon stiffness and modulus during tendon re-training after cast removal. In rats, 4 days of resistance training was found to induce AT tissue expression of LOX mRNA by up to 37-fold, indicating that training increases the degree of collagen cross-linking (Fratzl & SpringerLink ebooks - Engineering, 2008; Heinemeier et al., 2007). 1.7.4 Stiffness  Stiffness is a mechanical property of tendon and defined as change in tendon length in relation to the force applied to the tendon (Heinemeier & Kjaer, 2011). This parameter depends on the change of CSA and/or change in the Young’s modulus. Increases in tendon strength are frequently accompined with increase in tendon stiffness (Viidik, 1967). It has a significant influence on force transmission and muscle power (Bojsen-Moller, 2005). Therefore, increased tendon stiffness could also play an important role in the prevention of tendon injuries. Several studies have investigated the effect of different type of exercises on tendon stiffness. An increase in AT stiffness was found in healthy males after performing resistance execrcise of 70% of one repitition maximum of unilateral planter flexion (10 repitition/5sets, 8 weeks) (Kubo et al., 2002). Another study by Fouré et al., (2010) investigated the effect of plyometric training (jumping) on AT and found an increase in tendon stiffness after 14 weeks of training.   14 1.7.5 Young’s Modulus Young’s modulus (YM) is the intrinsic mechanical property of material, and is defined as the ratio between tendon stress and strain (Heinemeier & Kjaer, 2011). It mainly reflects the ability of material (i.e structure) to withstand load. Bayliss et al., (2016) looked at the effect of jumping and non-jumping legs of AT on YM. This cross-section study found that jumper had 24% greater YM than the non-jumper leg. Grosset et al., (2014) investigated the effect of high vs low intensity (40% of 1RM vs 80% of 1 RM) and only the group of high intensity demonstrated high YM.  1.8 Effect of Rest-Insertion Between Cyclic Load The effect of inserting rest periods during exercise has not been widely investigated in tendon, while several studies have found positive effects on bone formation. In a study by Srinivasan et al., (2002) using mice, inserting rest periods between cyclic loads (10s rest in low magnitude loading regimen of short bout of stretching) enhanced bone formation (osteogenesis) vs the control group (same magnitude loading regimen but with no rest). In addition, improved bone adaptation was found in a study using mice but with continuous high frequency vs 10 s rest between continuous high frequency cycles (LaMothe, 2004).  However, very few studies have investigated the effect of inserting rest periods during cyclic load in tendon. A recent study by Scott et al., (2011) using bioartificial tendon (mesenchymal cells) reported a greater tenocyte gene expression (SCX) and collagen I following cyclic vs static mechanical loading (5% strain) after 3 weeks in culture, and inserting a 10s rest period between each cycle potentiated the expression of SCX and collagen I. Another study by Huisman et al., (2014) investigated the effect of inserting 10s during cyclic load using primary fibroblasts derived from human hamstring tissue. There was a significant increase of gene expression for collagen I compared to continuous stimulated  15 tenocytes. All of these studies in both bone and tendon have demonstrated that rest interval that embedded into the exercise regimen could have substantial effect on tendon adaptation. 1.9 Effect of Immobilization on the Tendon Immobilization has harmful effects on all muscle-tendon unit components, causing atrophy of both muscle and tendon, but the effect on the tendon is slower and less dramatic (Jozsa, 1997). This can be explained by the tendon’s slower metabolic rate due to its poor vascularity and circulation, compared to muscle (Kannus, Józsa, Natri, & Järvinen, 1997). Overall, immobilization has a negative effect on tendons, causing them to eventually have a decreased tensile strength and elastic stiffness and a greater total weight (Jozsa, 1997). A number of experiments have focused on the effect of unloading/immobilization on the mechanical properties of tendons, and changes in the levels of collagen synthesis, but few experiments have explored changes in gene expression (Almeida-Silveira, Lambertz, Pérot, & Goubel, 2000; Matsumoto, Trudel,Uhthoff, & Backman, 2003; Reeves, Maganaris, Ferretti, & Narici, 2005). Both animal models and human experiments have shown that unloading/immobilization for periods ranging from 3 weeks to 3 months results in the reduction of collagenous tissue stiffness in the AT (Almeida-Silveira et al., 2000; Matsumoto et al., 2003; Reeves et al., 2005). In regards to changes that affect collagen fibrils due to immobilization, studies have shown a decrease in collagen fibril thickness in the rat AT (Nakagawa, Totsuka, Sato, Fukuda, & Hirota, 1989), reduction in collagen synthesis (Savolainen et al., 1988), and decrease in collagen content (Vailas et al., 1988). Also, in human AT after 15 weeks of immobilization, microscopically, collagen fibers become thinner, disoriented, and fragmented (Kannus et al.,1997). In addition, the cross links become reduced in number.   16 1.10 Effect of Aging on the Tendon With age, both collagenous and non-collagenous tissues are subject to modifications that alter the muscle architecture and tendon mechanical properties (Narici et al., 2008). Generally, aging combined with a sedentary life style is associated with a reduction in muscle mass, structure and strength, resulting in a dramatic decline in musculoskeletal performance, which profoundly affects quality of life (Seene & Kaasik, 2012). Both compositional and structural changes take place in tendons with aging, which are believed to cause alterations in the mechanical function. Collagen content may be reduced slightly, however, the relative amount of collagen content and the volume density decreases due to a reduction in the proteoglycan-water content with age (Kannus, 2000). In addition, an increase in collagen fiber diameter occurs with variations in thickness (Tuite, Renström, & O’brien, 1997). An increase in non-enzymatic cross-linking (glycation AGE) also occurs with aging, making the tissue stiffer and changing the normal matrix interaction leading to tissue dysfunction in the elderly (Bailey, 2001). The latter result was found experimentally in a study that examined collagen cross-linking densities of the patellar tendon in young and old males. The non-enzymatic-derived AGE marker (pentosidine) was elevated in older compared to younger males (Couppe et al., 2009). Because of the compositional changes, structural differences also appear with aging. One of the important structural parameters that changes with age is the tendon CSA, which has been shown in both animal and human studies (Nakagawa et al., 1989; Magnusson et al., 2003). In a recent study of the human AT, the CSA was larger in older men and women compared to younger subjects (Stenroth, Peltonen, Cronin, Sipilä, & Finni, 2012); the result was confirmed by previous work by Mangnusson et al., (2003) who found a larger AT CSA in older women compared to younger women. Increases in tendon CSA could be an  17 adaptation response, compensating for the loss of intrinsic material properties and to maintain tendon stiffness (Stenroth et al., 2012). 1.11 Effect of Sex Difference on Tendon Properties Evidence indicates that, on the whole, connective tissue injuries occur at a higher rate in women than in men (Bijur et al., 1997). Sex plays an important role in tendon adaptation and the discrepancy between males and females may be explained in part by the differences in sex hormones (Hewett, Myer, & Ford, 2006) . For instance, well-trained men (who have been running during the past 5 years) have patellar tendons with larger CSAs, compared to untrained men, but this difference was not observed when comparing trained and untrained women (Magnusson et al., 2007). Thus, men may adapt to physical loading activities with hypertrophy and increased collagen synthesis, response that is not found in women with similar activities, which may be related to sex differences and the effects of sex hormones such as estrogen or progesterone. Miller et al., (2005) measured the rate of collagen synthesis in both men and women at rest and after 72 hrs of performing a one-leg kicking exercise i.e rapid knee extension in seated position. Men had more collagen synthesis after exercise compared with women. In another study to understand whether or not estradiol had a direct effect on collagen synthesis in tendons at rest and after an acute bout of exercise, Hansen et al., (2008) measured a marker of collagen synthesis (PINP) in women who were taking oral contraceptives (OCs) (synthetic estradiol), compared to women who never used OCs. The authors found that women without OCs had an elevated collagen synthesis after exercise, whereas, women with OCs did not show any change in their collagen synthesis (Hansen et al., 2008) . Thus, the estrogen level can apparently influence the adaptation effect of acute exercises by reducing collagen synthesis.  18 1.12 Ultrasound Tissue Characterization Ultrasound tissue characterization (UTC) (Smartprobe 10L5; Terason 2000, Teratech, USA) is an imaging method that has been used to quantify tendon integrity in animals (Van schie, Bakker, Jonker, & Weeren, 2003) and humans (Van Schie et al., 2010). It captures contiguous transverse images (2D) at a regular distance (every 0.2 mm) along the tendon axis (12 to 20 cm). The images are transferred to the computer and compiled to create a 3D image for tomographic visualization in three planes: transverse, sagittal, and coronal. The stability of the pixel brightness from one transverse image to the next allows for conjectures to be made about the alignment of tendon bundles, and based on the UTC algorithm, the technology can quantify tendons into four echo-types (I–IV) (Van Schie et al., 2010). Based on validation studies conducted with equine tendon, type I echoes are mainly generated by intact and correctly aligned secondary tendon bundles; type II echoes are mainly generated by regions of tendon which demonstrate increased separation and/or more waving fascicles; type III echoes are generated by areas with decreased fibrillar integrity; and type IV echoes occur more commonly in tendon regions with a disorganized fibrillar structure or in regions with free fluid or amorphous material (de Vos, Weir, Tol, Weinans, & Verhaar, 2010). UTC was developed and introduced for the study of tendons in horses, and using tissue histology as the point of reference for comparisons to the corresponding ultrasonographic images, the echo pattern could be used to make calculated deductions about the underlying structure and/or pathology (Van schie, Bakker, Jonker, & Weeren, 2000; van Schie et al., 2003). Quantification of the transverse ultrasonographic images was first achieved by using a quantitative gray level statistic, though it was not sufficiently sensitive or accurate to quantify the homogeneity and axial alignment of tendon tissue (Van Schie et al., 2000). Subsequently, studies were conducted to compare the expressed ratios from UTC with  19 the histological appearance of the corresponding longitudinal tendon sections in vivo (Van Schie et al., 2003). The stability of the echo-pattern allowed for quantification by the variations in the gray level of each individual pixel in the adjacent transverse images (entropy), which is related to the degree of spatial homogeneity of the echo pattern. The result can be expressed by combining and correlating the entropy and waviness into four categories as described above, which are coded by color and superimposed onto the grey-scale ultrasound image. The four categories are coded by color as follows: Green (type I echoes) - pixels are characterized as steady with constantly high gray levels. Blue (type II echoes) - pixels have some characteristic of steadiness, with a gray level variation of approximately 10% or less. Red (type III echoes) - pixels have no characteristic of steadiness, with gray level variations >10%. Black (type IV echoes ) - pixels have no characteristic of correlation or entropy (van Schie et al., 2003). UTC differs from conventional imaging modalities (like standard US) by its ability to visualize and quantify integrity and by its potential to use a “hands-off” approach to scanning. In contrast, conventional US can be more easily influenced by manipulation of the ultrasound probe. Also, in theory, use of UTC can help eliminate anisotropy which found with conventional US. Anisotropy is an ultrasound artifact which present as decreased echogenicity of soft tissue; this is occur when ultrasound beam is not perpendicular to the imaged soft tissue long axis so the normal hyperechoic appearance is lost. This artifact may cause incorrect diagnosis as tendinopathy (Robinson, 2009).  In UTC, the tracking device prevents the transducer from tilting, and keeps it perpendicular to the tendon long axis to increase UTC repeatability (Van Schie et al., 2010).  UTC has been found to be a reliable research method to use in human subjects; with inter-observer and intra-observer reliability (intraclass correlation coefficient >0.92) (Van  20 Schie et al., 2010). It has also been validated to the extent that it consistently detects differences in echo-types between symptomatic and asymptomatic ATs (Van Schie et al., 2010). Echo-types I + II (structure-related) represent 51.5% of the total pixels in symptomatic tendons and 76.6% in the asymptomatic tendon, with an ICC of 0.92 (Van Schie et al., 2010). The ability of UTC to detect changes in echo-types was further supported by another study (Pollock, Antflick, & Smith, & Chakraverty, 2014). UTC was used to assess AT integrity differences between elite track and field athletes (male and females) and a significant difference was observed in the proportion of echo-types I, II, and IV for the asymptomatic males and females only. A significant difference was also found in echo-types III and IV between younger and older athletes >25 years, with older athletes displaying a greater proportion of type III and IV echoes, which could be interpreted as evidence of age-related degeneration or accumulated microinjuries (Pollock et al., 2014). Changes in echo-types were also found in an in vivo study where experimental and control groups of thoroughbred horses were evaluated for their response to maximal exercise in the superficial digital flexor tendon (Docking, Daffy, Schie, & Cook, 2012). The aim of the study was to detect the short-term tendon response to maximal exercises and the results showed a significant reduction in echo-types I and II (specifically on day 1 and 2) with no changes in the echotypes observed in the control group. Thus, there appears to be a short-term reduction in tendon morphology which is detectable by UTC. Although, there is structure similarities between human and horse’s tendon, the study might not be generalized to human (Patterson-Kane & Rich, 2014). Besides using UTC as a diagnostic tool to detect structural changes in tendons, UTC can also be used as a research tool to assess tendon structural integrity after rehabilitation interventions. Docking et al. (2015) found that the UTC echo-pattern improved in elite Australian football players during a five month pre-season period. A significant increase  21 occurred in echo-type 1 and a decrease occurred in echo-type II in pain-free ATs over the course of the five-month pre-season in elite male football players (Docking & Rosengarten, & Cook 2015). Also, UTC was used to monitor changes in AT structure after a platelet-rich plasma injection in patients with mid-portion Achilles tendinopathy (de Jonge et al., 2011). Although no significant changes were seen in the echo-types between the treatment and the placebo groups, UTC was able to detect small improvements in AT morphology of both groups over time. Echo-type I increased from 6 to 48 weeks, whereas echo-types II, III, and IV did not change over the same time. . In a recent study, amount of an aligned fibrillar structure (echo-types I + II) of normal and pathological Achilles and patellar tendons was measured with UTC (Docking & Cook, 2015). The main aim of the study was to investigate whether the improvement in tendon pain and function after certain types of exercises associated with improvement in tendon structure quantified by UTC. This cross-section study of Docking and Cook, 2015 reveals that pathological tendons (AT and patellar) contain a sufficient amount of echo type I + II. This is suggesting that pathological tendon might adapt by thickening to maintain a sufficient amount of echo-types (I + II) to tolerate the load without causing failure. (Docking & Cook, 2015). Although UTC has been validated in animals (horse tissue) with comparisons to histology, no equivalent data for UTC validation has been determined for human tissue. Previous research has also shown that UTC echo types can be correlated with the histomorphology of horse tendon tissue with different degrees of structural integrity (Van Schie et al., 2000; van Schie & Bakker, 2000; van Schie et al., 2003).  As a gold standard, UTC could be validated against surgery or MRI. Conventional US and high-resolution US has been commonly used to evaluate AT pathologies clinically and in published reports, because the procedure is non-invasive, quick, and cost-effective (Ellis, Teh, & Scott, 2002).  22 Several studies have also investigated the sensitivity and specificity of ultrasonography against criterion standards like MRI or surgery. Although little research has been carried out in this area, previous reports indicate that ultrasonography is a valuable tool for diagnosing Achilles tendinopathy  (Ibrahim & Elsaeed, 2013; Lehtinen, Peltokallio, & Taavitsainen, 1994). Therefore, UTC is a novel, reliable research technique that can be used to quantify tendon structure. From the above publications, UTC has been demonstarted to quantify subtle changes in tendon structure through four discriminated echo types. Based on the previous research, reductions in echo type I are associated with a reduction in the quality of tendon tissue which may signal tendon pathology. However, there is a lack of information related to alterations in echo types in response to physiologic loading, e.g. controlled isometric exercise. It is therefore possible that in different scenarios or situations the same echo type could result from different physiological or pathophysiological processes. 1.13 Objectives The overall objective of this study is to determine whether tendon morphology (determined using UTC) will improve following a high-magnitude, low-frequency isometric exercise program which includes either a short or long duration of rest period between each repetition (10s vs 3s). I hypothesized that ATs exposed to LRT would demonstrate increased tendon collagen organization and tendon cross-sectional area (CSA) compared with baseline, but that SRT would not demonstrate any change in either variable.   23 1.13.1 Aim of the Study.  To identify, using UTC, the impact of isometric exercise with varying durations of periodic rest on AT morphology and CSA before and after periods of resistance training. 1.13.2 Hypotheses.  H1: An increased percentage of type I echoes will be observed with LRT compared to baseline.  H10: An equivalent percentage of type I echoes will be observed with LRT compared to baseline.  H2: An increased CSA will be observed with LRT compared to baseline.  H20: An equivalent CSA will be observed with LRT compared to baseline. 1.14 Significance of the Research Appropriate mechanical loads are beneficial to tendons in that they enhance adaptation and promote anabolic processes (e.g., collagen synthesis and cross-linking, and improved material properties). Conversely, excessive mechanical loading promotes catabolic effects on tendons (e.g., matrix degradation and loss of material properties). Therefore, knowing the optimal characteristics of mechanical loading for tendons may have substantial benefits for maintaining tendon function, for the prevention of injury, and in the optimization of rehabilitation regimens.    24 Chapter 2. Methods 2.1 Participants The study was conducted in two cohorts and each cohort consisted of a 12 week training intervention. The first cohort involved 7 participants and the second cohorts involved 11 participants.  Thus, the study involved 18 healthy, recreationally active participants including 8 males and 10 females. This experimental study was conducted at the Center for Hip Health and Mobility (Vancouver Coastal Health Research Institute) and participants were recruited through posters and the investigators’ contact networks at the Center and at the University of British Columbia Vancouver campus. It took one month to recruit the participants before starting the study for both cohorts. Upon meeting with the potential participants, a letter of initial contact was given that introduced the researchers and explained the study. Once the participants expressed interest in enrolling in the study, they were given a consent form that further specifies the research protocol and their right to withdraw from the study. All 18 participants who were provided a consent form were eligible and agreed to participate, and no one was excluded from the study.  Inclusion criteria were: participant’s age was from 19 to 50 years; they were fluent in English, and they were self-described as recreationally active (i.e. neither completely sedentary, nor engaged in elite sporting activity). The exclusion criteria for the study were: participants living with a systemic inflammatory disease (e.g. Rheumatoid arthritis); possessing a neurological or musculoskeletal disorder that could be deemed to influence musculoskeletal properties (e.g., Marfan syndrome, cerebral palsy, hypermobility, etc); participants were also excluded if they had a recent (within 12 months) or current acute or chronic musculoskeletal disorder (e.g., tendinopathy, Haglund’s deformity, etc); participants with previous partial or full tear of AT, individual recently (within 12 months) participated in a structured lower resistance training program, or pregnant. The primary researcher Thuraya  25 Alketbi (TA) met all the recruited participants and to make sure that they read and understand all the experimental procedure and answer any question they have. In addition, the primary researcher (TA) was involved in conducting and instructing the participants for the training intervention for both cohorts. For the first cohort, Agnatha DeSa (AD) who was well trained in using UTC performed all the scans in the pre and post training intervention. In the second cohort, the primary researcher (TA) conducted all UTC scans for the pre and post training intervention. Patient data such as weight and height (used to obtain BMI), self-reported dominant leg, and age were collected at the first study visit and prior to the training intervention.  2.2 Randomization The right and left legs of each participant were randomly assigned to training interventions. Two sets of twenty sealed envelopes were generated, one for individuals who were right leg dominant, and one for individuals who were left leg dominant.  Each envelope contained a piece of paper indicating the allocation of the right and left legs to LRT or SRT. AS used a number generator to create the allocations using simple (unbalanced) randomization. The sealed envelopes were given to the primary researcher (TA) who kept them in a closed cabinet. A schedule was prepared ahead of the training intervention for each enrolled participants with time and date every week. Prior to training intervention, the primary researcher (TA) wrote the participant’s name on the front of the next available envelope based on participants dominant leg before opening the sealed envelope. Thus, after opening the sealed envelope and retrieving the allocation from inside, one leg was randomly allocated into the (LRT) group and the other leg was randomized into the (SRT) group. Eight participants were allocated to receive LRT on the dominant leg and 10 participants were allocated to receive SRT on the dominant leg (Table 2). All participants received the allocated training on both legs.  26 2.3 Blinding For the first cohort, Agnatha DeSa (AD), who was blinded to the type of the training allocated to each leg, performed all the scans before and after the training interventions. For the second cohort, the primary researcher (TA) conduct all UTC scans before and after the training intervention. For analysis of the scans (both cohorts), the primary researcher (TA) was blinded as to the time point (baseline or follow-up), participant’s identity and leg allocation. This blinding was achieved by another investigator (Alex Scott) who removed all identifying data from the UTC files and randomly assigned each scan a combination of alphabet letters followed by the number 1 or 2 (for first and second replication of each scan). It was not possible to blind the subjects or the investigators administering the exercise intervention to the exercise allocation. 2.4 Experimental Set-up 2.4.1 Pre-/Post-Training Prior to the training intervention the participants, anthropometric measurements (e.g., standing and seated height, foot and toe length, body mass, etc.) were taken prior to data collection. The International Physical Activity Questionnaire – Short Form (IPAQ-Short) was administered at the baseline (before the 12-weeks resistance training program). UTC of the AT was performed on all participants before the 12-week resistance-training program prior to the MVC testing and 24 hrs before the first training session. Post-training data collection (after 12 weeks of resistance training program) were IPAQ- short and UTC scan (24hr after last training session) (figure 4). Participants were asked to refrain from heavy exercise or ingesting anti-inflammatory drugs This was made clear for all participants before enrolling in the study and prior to the data collection. Before data collection (pre and post), the investigators verified verbally that these instructions had been followed, and all participants confirmed that they had followed these instructions.  27 2.4.2 Training Intervention The intervention for both LRT and SRT lasted 12 weeks and involved exercise performed three times a week. Both of the training interventions consisted of five sets of ten repetitive isometric contractions. The leg that was assigned to the LRT group had a 10s rest period inserted between each contraction (3s contraction, 10s relaxation), while the leg in the SRT group performed contractions without this rest (3s contraction, 3s relaxation). Both training groups had 60s rest between each set. LRT and SRT exercised at 90% of MVC, though to elicit ~5% tendon strain based on the experiments of Arampatzis et al. (2005). The repetitive isometric contractions (LRT or SRT) were used to induce cyclic strain on the triceps surae tendon and aponeurosis. Each participants were asked to perform several calf presses against the Biodex’s footplate for each leg, before starting the training session. All training was performed on isokinetic dynamometer (System 3, Biodex, New York, USA), a commonly used medical rehabilitation and research device to measure muscle force. The device is composed of a torque measurement unit (participants apply force through a footplate), an adjustable seat, and a control unit (PC unit). The participants can perform various types of exercises using specific muscle contraction types (isometric, concentric, and eccentric); in this study, all exercises were isometric. The participants were seated on the dynamometer chair, which was adjusted according to each participant’s anthropometric measurements. The internal hip, knee, and ankle angles were 95°, 180°, and 90°, respectively. The rotational axis of the dynamometer was aligned with the lateral malleolus of the fibula. To reduce any possible movement from the leg, upper body, or heel, straps were tightly applied over the foot and thigh of the exercised leg and participants were asked to cross their arms over their chest (Figure 5).  To obtain the peak torque of the planter-flexion, participants were asked to perform three isometric planter-flexor maximal voluntary contractions (MVC). Participants were  28 asked to warm up 2-3 minutes by performing few calf presses (submaximal isometric contraction) against the footplate of Biodex (figure 6). The foot was fixed at 90°; the participants produced 3-4s ramped MVC with a 1-minute rest between efforts. Strong verbal encouragement was given to the participants during each trial. Participants were asked to strongly push their pole of the foot against the footplate of Biodex machine and reach the target of their load as fast as possible. The tests were conducted on both legs. The plantar flexor MVC was tested at the beginning of each week and the training loads were modified accordingly. Participants were tested at the beginning of each week by performing maximum plantar flexion voluntary contraction, so based on that the training load (90% of the resultant MVC) were calculated and participants trained at 90% of the new MVC.  The training intervention was performed at the Center for Hip Health and Mobility (Vancouver Coastal Health Research Institute). The training was completed in the presence of the primary researcher (TA), Charlotte Waugh, or Alex Scott. A metronome/visual display from Biodex was used to achieve a consistent strain frequency and rate across the resistance training groups. Charlotte Waugh is a post doctoral fellow who was conducting a study of other parameters such as tendon stiffness and Young Modulus. The primary researcher (TA) is a physiotherapist who was trained to use Biodex during her practice.  Another volunteer (Bhav Kang, a kinesiologist who was trained in using the Biodex) assisted in participants training. A checklist and schedule was prepared to record each participant’s attendance. MVC and muscle torque data was automatically stored in the Biodex computer and retrieved at the end of the intervention for analysis. Adherence and compliance with the training intervention is presented in the results section. One modification was introduced in the second cohort, that each participants were asked to perform 30s of light jumping in place as a warm up.      29                Figure 4:  Experimental Set-Up  30 Figure 5: Biodex Setting.  2.5 Ultrasound Tissue Characterization (UTC) The UTC scans were obtained before and after the 12-week resistance training programs. Participants lay prone on the examination bed with their feet placed on a foot stabilizer so that the AT was perpendicular to the ultrasound transducer. To prevent any movement of the transducer while scanning it was held by a mechanical arm (with 360° of freedom) that was secured to a pole. To obtain measurement of echo-type proportions, the following parameters were used: window size 25, Entropy.Norm 8, Corr.Norm 120 and Coron Thres 120. 2.5.1 Measurement of the CSA. Through tomographic visualization of the ultrasound scans captured by the UTC device, the 3D image can be scrolled through and regions of interest can be visualized instantaneously in three planes of view (transverse, sagittal, and coronal plane), and a 3D rendered view is possible (Figure 6). Landmarks were determined 1 cm, 2 cm, and 3 cm from the calcaneal insertion (in the sagittal plane) along the tendon length, and the tendon border  31 was outlined (in the transverse plane). A representative image of the AT cross section is shown in Figure 6. ImageJ was used to measure the CSA. Test-retest reliability for the CSA at each location was measured as 1 cm, 2 cm, and 3 cm above the calcaneus insertion by the primary researcher (AT), and ICCs were found to be 0.83, 0.89, and 0.82, respectively. Figure 6: CSA of Achilles Tendon. The image on the right side represents the AT scanned with UTC software. The image on the left side is the AT CSA traced in ImageJ.  2.5.2 Measurement of Collagen Organization. UTC algorithms generate quantifications of echotypes from more highly ordered (type I) to less ordered (types II- IV), which have been demonstrated to relate, to some extent, to the architecture and integrity of the collagenous matrix. As described above, all images were de-identified to ensure the examiner was blinded during the analysis. The landmark was determined 2.5 cm from the calcaneal insertion in the sagittal plane and the tendon border was outlined (this outline is called a contour) at 10 scans proximal and 10 scans distal from the landmark. The two contours were interpolated to create a 4 mm (20 scans x 0.2mm) long region of interest for analysis. Proportions of the four echo types were calculated within the region of interest.    32 2.6 Validity and Reliability 2.6.1 Ultrasound Tissue Characterization (UTC). The inter-tester reliability of the UTC has been previously examined by van Schie et al. (2010), who reported an intraclass correlation coefficient (ICC) of 0.92 for Type 1 echoes. The reliability study for the UTC Type I echoes in the Scott laboratory was conducted by a different examiner (AD). The unpublished study was conducted in healthy women (n=9, 18 tendons). The interclass correlation coefficient (ICC) for echo type I was 0.90, and 0.87 for the sum of echo types II-IV. The minimum detectable change for the proportion of echo type I was 0.056. 2.6.2 Isokinetic Dynamometer. The isokinetic dynamometer was used to provide standardized body positions, torque, and velocity for repeated trials performed on the same day or on different days. The validity of the isometric torque and position measurements has been deemed as acceptable for both clinical and research purposes (Drouin et al., 2004). 2.6.3 IPAQ-Short Form. The IPAQ is a 7-day recall questionnaire that assesses frequency (days in the past week) and duration (minutes or/and hours a day) of walking, and moderate and vigorous intensity physical activity. The IPAQ short version takes about 5 minutes to complete and is scored with the Metabolic Equivalent of Task (MET) method for different activities and levels of intensities that are assigned different MET estimates. Responses are categorized into displaying a low, moderate, or high activity level. The criterion validity and reliability of the IPAQ-short form has been determined to be acceptable for different population i.e adult, youth and elderly (Lee, Macfarlane, Lam, & Stewart, 2011). The IPAQ-short is a questionnaire for use at the population level to monitor the level of physical activity or  33 inactivity and it has acceptable measurement properties; this questionnaire has been used in many different population and settings (Lee et al., 2011). 2.7 Data Analysis 2.7.1 Sample Size Based on a power analysis for the linear mixed effects model used in this study, the desired sample size for this study was 16 or a 0.058 change from baseline in the proportion of echo type I with 80% power. The sample size calculation was generated by a statistician using previous echo type I data from the Scott lab (AD reliability study).  2.7.2 Statistical Analysis. After consulting with a statistician, a linear mixed model (LMM) with alpha level set to 0.05 was used to examine the impact of the independent variables on two primary outcomes of interest (Type I echoes, and CSA), while also accounting for inter-individual variation and measurement variability between replicate scans. The independent variables were type of training (LRT vs. SRT) and time (baseline vs. 12 weeks). The linear mixed model was also used to determine whether or not there was a significant effect of baseline activity level, age, or sex. In addition to the main analysis, a secondary (exploratory) analysis was conducted using simple paired t tests (uncorrected) to compare the pre and post values of each echo-type (I, II, III, and IV), for the LRT legs and the SRT legs. UTC scans were made in duplicate for each participant at each time point, and both scans were analyzed and entered into the linear mixed model. All data were present and accounted for with the exception of the following: 4 participants dropped out of the study prior to completion, therefore their post-exercise data were not available. The statistician determined that the data met the assumptions of LMM. Prior to analysis, the data were visually inspected for outliers and for consistency from duplicate to duplicate – one error was detected (a transposition of digits during data entry) and corrected by pulling the original UTC scan. There were no extreme  34 outliers, so all the data were retained nothing removed. Baseline data for all 18 enrolled subjects were included in the model when examining the potential effects of covariates (activity level, age, sex) and for the estimates of sample variability. Four UTC scans could not be analyzed due to technical failure or artefact. The linear mixed model treated the four missing scans and the four drop out as missing data (missing at random; they were ignored, and no interpolation was carried out). Data are presented in the text as the mean (least square mean) and 95% confident interval (CI) as generated by the LMM analysis, with exception of participants’ beaseline characteristics and secondary analysis of individual echo-types, which are both presented as mean (SD). LS means are predictions from the linear mixed model or “unweighted mean” that are adjusted for unbalanced data. Visually, data are presented using box plots to denote the median, interquartiles, and range.     35 Chapter 3. Results 3.1 Participant Allocation and Drop-out The recruitment took one month for both cohorts. In total, there were four drop outs. One participant (female) dropped out in the first cohort after 10 weeks of training due to a complaint of pain while walking at the insertion of the left AT (Lt leg was allocated to SRT). In the second cohort, three participants had to drop out from the study; one female due to off-site injury (while playing rugby leading to a left ankle sprain). Another female participant developed temporary symptoms of overuse (soreness in the Achilles tendons and calf muscles bilaterally) due to the training session, after 6 weeks of training in both ankles, and voluntarily withdrew. These symptoms resolved fully after withdrawing from the program. The third participant, male suffered from sharp patellar pain (anterior left knee, leg was allocated to LRT) while performing an isometric contraction. All participants were contacted by AS and for all of them the pain disappeared after few weeks of rest. Figure 7 demonstrates a flow chart of the participants allocation and drop out.    36  Figure 7: Flow Diagram of Participant Allocations, Follow-Up, and Analyses.  Note, the analysis includes baseline data from 18 individuals, and post-intervention data from 14 individuals – see methods, statistics, for further details.  Table 1: Participant Characteristics; cm=centimetres, kg=kilograms. Values are Mean ± SD.  Dominant leg allocation Number Height (cm) Mass (kg) Age (years) Long rest training group (LRT) 8 171.08 (13.84) 68.70 (12.65) 32.00 (6.44) Short rest training group (SRT) 10 174.39 (10.91) 71.24 (13.35) 27.30 (5.60)     37 3.2 Compliance  With regard to the number of training sessions that were attended, the average  attendance was 95% (on average, 34 of 36 sessions were attended). Attendance ranged from 32 to 35 sessions, i.e.  no participant missed more than 4 training sessions throughout the training period. Participants were instructed to train at 90% of their MVC, but there was some variation due to inability to precisely reach this target. This variability was assessed on the first and last sessions by retrieving data from the Biodex and calculating the average %MVC of all 50 iosmetric contractions during these sessions. On the first training session, participants tended to overshoot their target – average training loads (calculated for the entire session, i.e. all five sets) were 111% (12%) of MVC for LRT and 108% (16%) of MVC for SRT. At the last training session, the average training loads for the LRT and SRT legs during training were 90% (13%) for LRT and 81% (7%) for SRT. This indicates that there may have been more fatigue in the SRT group, causing them to fall short of the prescribed training load. 3.3 Participants Physical Activity   Based on the (IPAQ) short scoring classification criteria, 13 of the participants were categorized as having a high activity level and a few participants had activity levels that were moderate (n=2) or low (n=3). All of the participants but two stayed at the same level of activity before and after the 12-week period of resistance exercise (Table 2); one subject incerased from low to moderate levels, and one decreased from moderate to low. An exercise diary was also used to monitor the participants’ daily activities. The activities in the ITR group included yoga, skiing, hockey, soccer, running, upper limb weight training, basketball, spinning, cardio workouts, bicycling, and volleyball. The activities for the SRT group included jogging, walking, soccer, squash, Frisbee, basketball, volleyball, climbing stairs, upper limb weight training, spinning, dragon boat paddling, bicycling, and tennis (see appendix A&B).  38 Table 2: Participant Activity Level Based on IPAQ Classifications. Pre-training Post-training MET score n MET score n High ≥ 3000 MET.min.wk-1 13 High ≥ 3000 MET.min.wk-1 11 Moderate  ≥ 600 MET.min.wk-1 2 Moderate ≥ 600 MET.min.wk-1 1 Low < 600 MET.min.wk-1 3 Low < 600 MET.min.wk-1 2 3.4 Type I Echoes The proportion of echo-type I for the AT at baseline was 0.60 (95% CI = 0.56 to 0.65) in the ATs assigned to the LRT training and 0.66 (95% CI= 0.60 to 0.70) in the ATs assigned to the SRT training (Figure 8, A). After 12 weeks, a statistically significant reduction was observed in the proportion of echo-type I for the AT in the SRT tendons 0.55 (95% CI= 0.49 to 0.60, p < 0.05). No statistically different change was observed in the proportion of echo-type I for the AT in the LRT tendons 0.62 (95% CI= 0.57 to 0.67). A statistically significant interaction was observed between the presence of low baseline physical activity level and the occurrence of a post-exercise reduction of type I echoes (Figure 8, B). However, only two participants for whom post-intervention data was available were categorized as having a low activity level at baseline, so this result must be interpreted with caution. Neither age nor sex had a significant influence on the proportion of type I echoes.  39  Figure 8: Effect of Resistance Exercises on Type I Echoes. UTC scans were taken before (pre) and after (post) 12 weeks of resistance training exercise. (SRT) represents short rest training, (LRT) represents long rest training. The bars represent the range of values between the 25th and 75th percentiles. The horizontal line inside the bar is the median, and the whiskers represent minimums and maximums in the dataset, except where the individual outliers are shown by open circles.    40 3.5 Cross-Sectional Area No appreciable differences were seen in the CSA of the ATs assigned to the two types of training (LRT vs. SRT) or over time (baseline vs. 12 weeks) (Table 3). No statistically significant changes over time were observed at any of the three locations (Figure 9). Similarly, age and level of activity did not show any significant influence on the CSA of the AT in either of the training (LRT or SRT) groups. A statistically significant interaction between tendon CSA and both sex and moderate level of activity (p < 0.05) were observed at baseline. However, only two participants were categorized as having a moderate activity level, so this result must be interpreted with caution. There was a statistically significant difference on tendon CSA between both moderate and low activity level (p < 0.05), however the 95% CIs were overlapping. Table 3: CSA Measurements Before and After 12 Weeks of Resistance Training; Presented in cm as LS Means (95% CI). CSA Before Training After Training Number  Location    1     2    3  Type of exercise    SRT              LRT  Sex    Male     Female  Activity level    Low    Moderate    High  n= 18  0.62 (0.54 to 0.68) 0.57 (0.50 to 0.64) 0.55 (0.47 to 0.61)   0.56 (0.49 to 0.63) 0.59 (0.52 to 0.66)    0.70 (0.63 to 0.76)*  0.48 (0.42 to 0.54)*      0.49 (0.33 to 0.65)*    0.47 (0.28 to 0.67)*    0.61 (0.53 to 0.69) n=14  0.62 (0.55 to 0.69) 0.58 (0.51 to 0.64) 0.53 (0.46 to 0.60)   0.58 (0.50 to 0.64) 0.57 (0.50 to 0.64)   0.69 (0.62 to 0.75) 0.49 (0.43 to 0.55)   0.45 (0.29 to 0.61) 0.50 (0.30 to 0.70) 0.61 (0.54 to 0.69)   41  Figure 9: Effect of Resistance Exercise on the Cross-Sectional Area of the Achilles Tendon. UTC scans were taken before (pre) and after (post) 12 weeks of resistance training exercise. (SRT) represents short rest training, (LRT) represents long rest training. Location (1) is above the Achilles tendon insertion (1 cm), location (2) is above the Achilles tendon insertion (2 cm), and location (3) is above the Achilles tendon insertion (3 cm). The bars represent the range of values between the 25th and 75th percentiles. The horizontal line inside the bar is the median, and the whiskers represent minimums and maximums in the dataset. No statistically significant difference was observed after 12 weeks of training at any of the locations (1, 2, or 3).     42 3.6 Muscle Strength  As shown in Figure 10, muscle torque increased for both types of training exercise (LRT and SRT). Because strength is displayed as an improvement from baseline, there was no necessity to account for the moment arm, which varied from person to person but is assumed to remain constant over the duration of the study. At baseline, the average torque (non-normalized for moment arm) was 149.6 (50.1) N.m for the LRT and 151.9 (38.3) N.m for SRT. Following the 12 weeks of training, torque was 212.8 (68.0) N.m and 218.6 (42.1) N.m for LRT and SRT, respectively.   Figure 10: Muscle Strength (torque) Percentage of Change from Baseline According to Type of Training. Muscle strength was measured  after 12 weeks of resistance training exercise. The bars represent ranges of values between the 25th and 75th percentiles. The horizontal line inside the bar is the median, and the whiskers represent minimums and maximums in the dataset.  0.0050.00100.00150.00200.00250.00LRT SRT% Change from baselineMuscle strength change after training 43 3.7 Effect of Long Rest Exercise on Echo-Pattern No significant changes in the proportion of echo types were observed over time (baseline vs. 12 weeks) (Figure 11).   Figure 11: Effect of Long Rest Training on Ultrasound Tissue Characterization (Proportion of Echo-Types). The bars show the proportion of echo-types. The error bars show the mean and SD. No significant change in the proportion of echo types I, II, III, and IV was observed after the training exercise.    0.000.100.200.300.400.500.600.700.800.90Pre Post Pre post pre post pre postEchotype I Echotype II Echotype III Echotype IVproportion of echo typeMean Echotypes (+/- 1 SD)Long Rest Training 44 3.8 Effect of Short Rest Exercise on Echo Pattern As mentioned previously, a significant reduction in the echo type I was observed in the SRT group. Therefore, we conducted an exploratory analysis to determine whether other individual echo types were altered.  A significant increase in the proportion of echo type II was observed after the training period 0.403 (0.153) compared to the baseline 0.304 (0.066), p < 0.05) (Figure 11). Similarly, a small but statistically significant increase in the echo type III was also seen after the training period 0.036 (0.041), compared to the baseline (0.018, (0.018) (p < 0.05). No significant difference was observed in the echo type IV.   Figure 12: Effect of Short Rest Exercise on Tissue Characterization (Echo-Types). The bars show the proportion of the echo types. The error bars show the mean and SD. A significant reduction occurred in Type I echoes, and there was a corresponding increase in Type II and III echoes after the training period.     0.000.100.200.300.400.500.600.700.80Pre Post Pre post pre post pre postEchotype I Echotype II Echotype III Echotype IVproportion of echo typeMean Echotypes (+/- 1 SD)Short Rest Training 45 Chapter 4. Discussion In this study, we found that high strain, low frequency, isometric plantarflexion training resulted in a significant reduction in type I echoes in the AT. This reduction was specifically observed in the tendons that were allocated to the SRT, but was not observed in tendons allocated to receive the same exercise but with the insertion of a 10s rest period between loading repetitions (LRT). The reduction in type I echoes observed in the SRT suggests that there was a relative decrease in the intact and aligned secondary collagen bundles (fibers and fascicles) in response to this type of training. We did not see any evidence of increased CSA for the AT with either type of exercise after the 12-week training period. 4.1 Type I Echoes Alteration in SRT Type I echoes are generated by the UTC algorithm in regions of the tendon which demonstrate high stability in the grey scale level of pixels over multiple transverse scans, which is interpreted to represent predominantly intact and aligned tendon bundles (Van Schie et al., 2010). A reduction in the proportion of type I echoes over the 12-week training period in the SRT group therefore suggests a decrease in the proportion of tendon matrix which is composed of intact and aligned collagen fiber bundles. This reduction in echo type I could make the tendon more vulnerable to injury such as tendinopathy.  However, whether or not the decrease in the proportion of Type I echoes is due to reductions in the relative amount of highly organized fibrillar collagen or rather to an increase in the relative amount of non-collagenous matrix is unknown. Changes in proteoglycan (PG) accumulation and glycosaminoglycans (GAGs) have been found with exercise (Hae Yoonet al.2003). Proteoglycan production is integral to the process of collagen fibrillogenesis, and PGs contribute in and of themselves to the structure and mechanical properties of tendons (Yoon & Halper, 2005). Yoon et al. (2003) found increased levels of PG (decorin) and  46 associated GAGs after 4 weeks of daily running for 30 minutes in avian gastrocnemius tendons. The increased production of PG and GAGs (water-binding protein) may lead to increased amounts of ground substance in tendons (Screen et al., 2005). This is one way in which tendons may undergo local changes in structure without changing the biomechanical integrity of the tendon. However, arguing against this line of reasoning is the fact that there was no change in tendon CSA following exercise. If PG levels were increased, this might have been expected to raise the water content level of the tendon and result in increased CSA. In addition, taking into consideration that the Yoon et al., (2003) is an animal study, this line of thinking is speculative.  4.2 Alteration in Type II and III Echoes? The secondary (exploratory) analysis suggested that the decrease in the proportion of type I echoes in the SRT group coincided with an increase in type II and III echoes, suggesting a subtle change in tendon organization. However, the physiological or pathophysiological changes underlying this morphological change are unknown. According to Van Schie et al.,( 2010) type II echoes indicate the presence of discontinuous or wavy fibers and fascicles, and type III echoes indicate the presence of loose, fibrillar matrix (Van Schie et al., 2010). Albeit speculative, we could suggest that perhaps no collagenous damage occurred in the SRT group, but rather that there was an increased production of local inflammatory mediators and possibly an increase in tendon vascularity. This thinking is based on the continuum model of tendon pathology developed by Cook and Purdam  (Cook & Purdam, 2009) . In particular, the early stages of tendon overuse (“reactive tendinopathy”) are thought to be characterized by a proliferative tissue reaction which may initially be asymptomatic (McCreesh & Lewis, 2013). The second stage is tendon disrepair, which is manifested as matrix breakdown, collagen separation, and neovascularity (McCreesh & Lewis, 2013). According to the continuum model, the first two stages may be reversible,  47 which could explain the changes seen in the echo types in the SRT group that manifested by the presence discontinuous or wavy fibers and fascicles due to increased cellular response with no collagenous damage.  However, there is a lack of literature that supporting the continuum model of tendon pathology in humans and its relation to UTC morphology.  4.3 Alteration in Echo Types as An Adaptive Response? In an animal study, Docking et al.,  (2012) investigated changes in the structure of the superficial digital flexor tendon (SDFT) in thoroughbred horses following maximal exercise. UTC scans were taken before the race and on day 1, 2, and 3 post-race. Changes in the echo pattern were found on days 1 and 2, which returned to baseline on day 3. A significant reduction was observed in type I echoes and corresponding increases were observed in type II and III echoes on days 1 and 2 post-race. Interestingly, the echo pattern returned to baseline after 72 hours (day 3) post-race.  In another study, a microdialysis technique was used in the AT peri-tendon before and after (120 min) and 72 h of 36 km of running (Langberg et al., 1999). The authors found that exercise induced changes in metabolic and inflammatory activity immediately after running. Nevertheless, a reduction in PICP (collagen I marker) occurred immediately after running, but increased 3-fold after 72 h, indicating that acute exercise induced tendon changes leading to increased formation of collagen I during the recovery periods. Taken together, these findings indicate that the recovery periods may be needed for collagen I synthesis and the clearing out of GAGs, and metabolic and inflammatory mediators. Perhaps the UTC changes observed in the SRT group in this study are subtle morphological reflections of an active adaptation response occurring during the acute post-exercise recovery period. However, the loads in the latter studies were different (sports loading vs isometric load).  48 4.4 Long Rest Insertion and Changes in Echo Type I  In the LRT tendons, no reduction in the proportion of type I echoes was observed, which was in contrast to the reduction of type I echoes observed in the SRT tendons. The reason for this difference in the morphological appearance associated with these two types of training is not known. In an in vitro study by Huisman et al. (2014) using human hamstring tenocytes, the insertion of a 10s rest period between the cyclic loads resulted in increased production of collagen type I mRNA and protein, an effect which was not observed with continuous loading (Huisman et al., 2014). This suggests that perhaps the rest insertion between the cyclic loads provided adequate time for recovery, which may facilitate the adaptive response. In this line of thinking, the morphological changes seen in the SRT group would represent features associated with the beginnings of overuse injury resulting from inadequate adaptation, whereas these changes were avoided through the insertion of rest-periods in the LRT group. Perhaps the LRT loading regimen was more capable of inducing an adaptive response capable of allowing the tendon to resist the development of subtle microinjuries. However, given that the study of Huisiman et al., (2014) is a cell culture study, this line of thinking is very speculative.   4.5 CSA and Mechanical Load  We found no significant difference in the CSA of tendons subjected to either type of training. This is supported by research where an increased tendon CSA occurred only with more chronic exposure to repetitive loads (Magnusson & Kjaer, 2003; Magnusson et al., 2001). In a study by Arampatzis et al. (2007), the authors found regional differences in the AT CSA at 60-70% of the tendon length after 14 weeks (4 times per week) of cyclic and high magnitude resistance training. In this study; however, measurements of the AT CSA were taken at several locations along the length of the tendon and training was 3 times per week for 12 weeks which may attributed to not see any differences in the tendon CSA after either  49 type of training. Additionally, in the Arapmatzis et al., (2007), MRI was used to measure CSA as opposed to the current study which used transverse ultrasound scans. 4.6 Did LRT Provide Sufficient Stimulus to Cause AT Adaptation? We hypothesized that LRT would cause an increase in the extent of tendon collagen organization, but we found no such change in either of the two groups. In this study, we used a high-strain intervention that was comparable to the exercise protocol used by Arampatzis et al., (2007), which was indeed found to be sufficient to trigger an anabolic response and cause an increase in tendon-aponeurosis stiffness, elastic modulus, and region-specific hypertrophy of the AT. When we examined training load, we found that some participants were not able to exercise the leg at 90% of their MVC for the entire duration of the training sessions. However, this was the case both for LRT and SRT tendons, therefore lack of sufficient training stimulus in the LRT group is not a likely explanation for the rejection of the original hypothesis. 4.7 Missing Data Four participants dropped out of the study. Two dropped out due to overuse symptoms, one had sharp pain during the isometric training and the one had an ankle sprain (off-site injury). The participants who dropped out of the study seem similar to those who remain, although based on the small number of drop-outs (n=4) and completers (n=14), it is difficult to make a systematic comparison (for individual data, please see Appendices). The dropped out participants included three females, one with a high activity level (off site injury) and two with low activity levels (overuse symptoms which resolved over several weeks). The last participant who dropped out was male (sharp patellar pain during isometric exercise which resolved over several weeks). The LMM treated participant’s data (post exercise) as missing, with the assumption being that it was missing at random. This assumption is  50 debatable, given that two participants who dropped out due to overuse symptoms had a low physical activity, particularly given that the linear mixed model found an association between low activity level and a post-exercise reduction of Type I echoes. The decision was made to include all available data in order to (a) retain as much data as possible for analysis thereby improving statistically power, and (b) avoid introducing bias into the analysis by discarding data from non-completers.  4.8 Limitations In this study, sample size was a limitation. In future studies, a larger number of participants with varying starting activity levels could strengthen the findings for both of the exercise groups and clarify the effect of rest duration on adaptation of AT collagen organization. A potential limitation of our study is that we examined the tendons with UTC within 48 hours of the last exercise session. Thus, acute post-exercise changes may have contributed to the observed changes, rather than morphology attributed to adaptations occurring over the 12-week training period. Given that the reduction of Type I echoes was specifically seen in the SRT, as opposed to the LRT, group, a future study could examine the potential short-term response of echo types to these two types of training. Moreover, this study was hampered by the lack of reference studies for comparison. Few authors have used UTC in healthy tendons, and typically, such studies used different positions for scanning (lying down vs. standing), different UTC components (manual tracking vs. automatic), different ROIs across the tendon, and variations on the algorithm used to generate echo types (e.g. varying number of adjacent scans over which pixel stability / entropy is calculated, a.k.a. “window size”).  51 4.9 Future Directions Since some changes were observed in the UTC echo patterns of the AT, further research could explore whether the changes were due to anabolic or catabolic processes or a combination of both. Other techniques, like microdialysis, could be used in combination with UTC to further our understanding of collagen synthesis and degradation, and tendon structural adaptation. By measuring the mechanical properties of tendons, like stiffness and the Young modulus, we can also gain a better understanding of the impact on tendon function and whether the tendon overall experienced an improvement or a reduction in tensile properties.  52 Chapter 5. Conclusion The results of this study demonstrate a decrease in the relative proportion of type I echoes in the AT after 12 weeks of SRT (short rest training), and no change in tendon morphology after LRT (long rest training). The CSA of the AT was not different after 12 weeks of high magnitude resistance training for either type of training. Due to the various issues discussed above, alternative explanations centring either on injury or on adaptation could be posed to account for these findings, therefore our current ability to interpret the significance of these morphological changes is quite limited. Further data analysis from this group of individuals is ongoing, including Young’s modulus, which will provide more context in which to understand the altered morphology. (Young’s modulus is the ratio of stress/strain, and indicates a change in composition of the tendon microstructure such as improved or decreased collagen arrangement).  In addition, a future study with long-term follow-up may help in identifying whether the changes seen with UTC are associated with tendon adaptation or injury.    53 References Almeida-Silveira, M., Lambertz, D., Pérot, C., & Goubel, F. (2000). Changes in stiffness induced by hindlimb suspension in rat Achilles tendon. 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Clinical Biomechanics, 16(3), 245-251. doi:10.1016/s0268-0033(00)00089-9     67 Appendix A: International Physical Activity Questionnaire Form We are interested in finding out about the kinds of physical activities that people do as part of their everyday lives. The questions will ask you about the time you spent being physically active in the last 7 days. Please answer each question even if you do not consider yourself to be an active person. Please think about the activities you do at work, as part of your house and yard work, to get from place to place, and in your spare time for recreation, exercise or sport. Think about all the vigorous activities that you did in the last 7 days. Vigorous physical activities refer to activities that take hard physical effort and make you breathe much harder than normal. Think only about those physical activities that you did for at least 10 minutes at a time. 1. During the last 7 days, on how many days did you do vigorous physical activities like heavy lifting, digging, aerobics, or fast bicycling? _____ days per week  No vigorous physical activities   Skip to question 3  2. How much time did you usually spend doing vigorous physical activities on one of those days? _____ hours per day _____ minutes per day  Don’t know/Not sure  Think about all the moderate activities that you did in the last 7 days. Moderate activities refer to activities that take moderate physical effort and make you breathe  68 somewhat harder than normal. Think only about those physical activities that you did for at least 10 minutes at a time. 3. During the last 7 days, on how many days did you do moderate physical activities like carrying light loads, bicycling at a regular pace, or doubles tennis? Do not include walking. _____ days per week  No moderate physical activities   Skip to question 5  4. How much time did you usually spend doing moderate physical activities on one of those days? _____ hours per day _____ minutes per day  Don’t know/Not sure  Think about the time you spent walking in the last 7 days. This includes at work and at home, walking to travel from place to place, and any other walking that you have done solely for recreation, sport, exercise, or leisure. 5. During the last 7 days, on how many days did you walk for at least 10 minutes at a time? _____ days per week  No walking     Skip to question 7  6. How much time did you usually spend walking on one of those days? _____ hours per day _____ minutes per day  69  Don’t know/Not sure  The last question is about the time you spent sitting on weekdays during the last 7 days. Include time spent at work, at home, while doing course work and during leisure time. This may include time spent sitting at a desk, visiting friends, reading, or sitting or lying down to watch television.  7. During the last 7 days, how much time did you spend sitting on a week day? _____ hours per day _____ minutes per day  Don’t know/Not sure   This is the end of the questionnaire, thank you for participating.   70 Appendix B: Participant Characteristics and Activities LRT group Participant Sex Age Side Dominant Rt Side Allocation Height/cm Weight/kg Activities         1 M 24 Rt LRT 186 78 Yoga, skiing, hockey 2 M 23 Rt LRT 177 75 Basketball, strengthening &conditioning, cardio 3 M 35 Rt LRT 183 82 running, spinning, stepper, bicycling 4 M 43 Rt LRT 180.8 80.9 soccer, running 5 F 34 Rt LRT 153.3 53.2 walking 6 F 31 Rt LRT 149.2 58 walking 7 F 35 Lt LRT 164.8 51.2 walking 8 F 31 Rt LRT 174.5 71.3 bike, weight training, volleyball, hiking, tennis, running   SRT group Participant Sex Age Side Dominant Rt Side Allocation Height/cm Weight/kg Activities         9 F 27 Rt SRT 171 57 jogging, walking 10 F 23 Rt SRT 161.8 61 ultimate league, soccer 11 F 27 Rt SRT 173 69 squash, spin, upper limb weight training, bike, 12 F 30 Rt SRT 160 63.5 dragon boat paddling exercises, climbing, Frisbee, walking, upper limb weight training 13 F 37 Rt SRT 163 55 walking, climbing stairs, spin classes, stationary bike, bicycling  71 14 F 21 Rt SRT 172.7 63.5 running, upper limb weight training upper &lower body, basketball, tennis, volleyball, 15 M 36 Rt SRT 184.4 91.4 walking 16 M 27 Rt SRT 180 86 Upper limb weight training, soccer, judo 17 M 23 Rt SRT 188 81 Upper limb weight training for upper &lower body, cardio 18 M 22 Rt SRT 190 85 swimming   72 Appendix C: IPAQ Analysis Result  Participant Sex Age IPAQ score at baseline Category IPAQ score after 12 weeks Category 1 M 24 5106 High 11892 High 2 M 23 5253 High 5706 High 3 M 35 6926 High 7019 High 4 M 43 1899 High 3798 High 5 F 34 1455 Moderate 615 Low 6 F 31 unknown High 2856.0 High 7 F 35 unknown Low dropout - 8 F 31 4239 High 3786 High 9 F 27 2079.0 Moderate - - 10 F 23 5253.0 High - - 11 F 27 5287.5 High 7890.0 High 12 F 30 3439.5 High 4053.0 High 13 F 37 1386.0 low 2004.0 Moderate 14 F 21 2598.0 High 3546.0 High 15 M 36 132.2 Low 99.0 Low 16 M 27 4953.0 High 4293.0 High 17 M 23 7278.0 High 8853.0 High 18 M 22 3546.0 High - - (-) drop out    73 Appendix D: Echo-Types Measurements proportion of Type 1 echoes Participant Activity level Age Sex Cohort Rest Leg Dominant leg Pre/Post 0.61 1 high 43 m 1 y r y pre 0.64 1 high 43 m 1 y r y pre 0.69 1 high 43 m 1 y r y post 0.78 1 high 43 m 1 y r y post 0.69 1 high 43 m 1 n l n pre 0.68 1 high 43 m 1 n l n pre 0.65 1 high 43 m 1 n l n post 0.62 1 high 43 m 1 n l n post 0.57 2 high 31 f 1 y r y pre 0.61 2 high 31 f 1 y r y pre 0.7 2 high 31 f 1 y r y post 0.73 2 high 31 f 1 y r y post 0.59 2 high 31 f 1 n l n pre 0.62 2 high 31 f 1 n l n pre 0.61 2 high 31 f 1 n l n post 0.6 2 high 31 f 1 n l n post 0.74 3 high 31 f 1 y r y pre 0.73 3 high 31 f 1 y r y pre 0.76 3 high 31 f 1 y r y post 0.72 3 high 31 f 1 y r y post 0.66 3 high 31 f 1 n l n pre 0.7 3 high 31 f 1 n l n pre 0.69 3 high 31 f 1 n l n post 0.63 3 high 31 f 1 n l n post  74 0.74 4 low 36 m 1 n r y pre 0.74 4 low 36 m 1 n r y pre 0.68 4 low 36 m 1 n r y post 0.74 4 low 36 m 1 n r y post 0.63 4 low 36 m 1 y l n pre 0.62 4 low 36 m 1 y l n pre 0.8 4 low 36 m 1 y l n post 0.72 4 low 36 m 1 y l n post 0.5 5 moderate 34 f 1 y r y pre 0.56 5 moderate 34 f 1 y r y pre 0.65 5 moderate 34 f 1 y r y post 0.66 5 moderate 34 f 1 y r y post 0.52 5 moderate 34 f 1 n l n pre 0.53 5 moderate 34 f 1 n l n pre 0.4 5 moderate 34 f 1 n l n post 0.45 5 moderate 34 f 1 n l n post 0.77 6 high 35 m 2 y r y pre 0.79 6 high 35 m 2 y r y pre 0.72 6 high 35 m 2 y r y post 0.73 6 high 35 m 2 y r y post 0.7 6 high 35 m 2 n l n pre 0.74 6 high 35 m 2 n l n pre 0.49 6 high 35 m 2 n l n post 0.51 6 high 35 m 2 n l n post 0.39 7 high 23 f 2 y r y pre 0.39 7 high 23 f 2 y r y pre 0.38 7 high 23 f 2 n l n pre 0.78 8 high 23 m 2 n r y pre 0.76 8 high 23 m 2 n r y pre  75 0.76 8 high 23 m 2 n r y post 0.74 8 high 23 m 2 n r y post 0.65 8 high 23 m 2 y l n pre 0.66 8 high 23 m 2 y l n pre 0.57 8 high 23 m 2 y l n post 0.53 8 high 23 m 2 y l n post 0.69 9 high 22 m 2 n r y pre 0.72 9 high 22 m 2 n r y pre 0.51 9 high 22 m 2 y l n pre 0.56 9 high 22 m 2 y l n pre 0.62 10 high 24 m 2 y r y pre 0.52 10 high 24 m 2 y r y pre 0.52 10 high 24 m 2 y r y post 0.75 10 high 24 m 2 y r y post 0.66 10 high 24 m 2 n l n pre 0.66 10 high 24 m 2 n l n pre 0.71 10 high 24 m 2 n l n post 0.71 11 low 37 f 2 n r y pre 0.72 11 low 37 f 2 n r y pre 0.2 11 low 37 f 2 n r y post 0.18 11 low 37 f 2 n r y post 0.67 11 low 37 f 2 y l n pre 0.67 11 low 37 f 2 y l n pre 0.53 11 low 37 f 2 y l n post 0.26 11 low 37 f 2 y l n post 0.76 12 moderate 27 f 2 n r y pre 0.65 12 moderate 27 f 2 n r y pre 0.52 12 moderate 27 f 2 y l n pre 0.68 12 moderate 27 f 2 y l n pre  76 0.73 13 high 23 m 2 y r y pre 0.66 13 high 23 m 2 y r y pre 0.67 13 high 23 m 2 y r y post 0.69 13 high 23 m 2 y r y post 0.64 13 high 23 m 2 n l n pre 0.64 13 high 23 m 2 n l n pre 0.39 13 high 23 m 2 n l n post 0.41 13 high 23 m 2 n l n post 0.54 14 high 27 m 2 y r y pre 0.64 14 high 27 m 2 y r y pre 0.6 14 high 27 m 2 y r y post 0.55 14 high 27 m 2 y r y post 0.78 14 high 27 m 2 n l n pre 0.78 14 high 27 m 2 n l n pre 0.51 14 high 27 m 2 n l n post 0.57 14 high 27 m 2 n l n post 0.56 15 high 30 f 2 n r y pre 0.56 15 high 30 f 2 n r y pre 0.47 15 high 30 f 2 n r y post 0.55 15 high 30 f 2 n r y post 0.55 15 high 30 f 2 y l n pre 0.54 15 high 30 f 2 y l n pre 0.27 15 high 30 f 2 y l n post 0.3 15 high 30 f 2 y l n post 0.49 16 high 27 f 2 n r y pre 0.44 16 high 27 f 2 n r y pre 0.54 16 high 27 f 2 n r y post 0.69 16 high 27 f 2 n r y post 0.61 16 high 27 f 2 y l n pre  77 0.77 16 high 27 f 2 y l n post 0.62 16 high 27 f 2 y l n post 0.63 17 low 35 f 1 y r n pre 0.62 17 low 35 f 1 y r n pre 0.76 17 low 35 f 1 n l y pre 0.72 17 low 35 f 1 n l y pre 0.61 18 high 21 f 1 y l n pre 0.62 18 high 21 f 1 y l n pre 0.72 18 high 21 f 1 y l n post 0.69 18 high 21 f 1 y l n post 0.67 18 high 21 f 1 n r y pre 0.69 18 high 21 f 1 n r y pre 0.72 18 high 21 f 1 n r y post    78 Appendix E: CSA Measurements Location CSA Participant Activity level Age Sex Cohort Rest Leg Dominant leg Pre/Post 1 0.851 1 high 43 m 1 y r y pre 1 1.012 1 high 43 m 1 y r y pre 1 0.973 1 high 43 m 1 y r y post 1 0.975 1 high 43 m 1 y r y post 2 1.021 1 high 43 m 1 y r y pre 2 1.046 1 high 43 m 1 y r y pre 2 0.813 1 high 43 m 1 y r y post 2 0.962 1 high 43 m 1 y r y post 3 0.879 1 high 43 m 1 y r y pre  3 1.07 1 high 43 m 1 y r y pre 3 0.841 1 high 43 m 1 y r y post 3 0.837 1 high 43 m 1 y r y post 1 0.836 1 high 43 m 1 n l n pre 1 0.755 1 high 43 m 1 n l n pre 1 0.908 1 high 43 m 1 n l n post 1 0.87 1 high 43 m 1 n l n post 2 0.851 1 high 43 m 1 n l n pre 2 0.862 1 high 43 m 1 n l n pre 2 0.811 1 high 43 m 1 n l n post 2 0.927 1 high 43 m 1 n l n post 3 0.74 1 high 43 m 1 n l n pre  3 0.819 1 high 43 m 1 n l n pre 3 0.86 1 high 43 m 1 n l n post 3 0.706 1 high 43 m 1 n l n post 1 0.923 2 high 31 f 1 y r y pre 1 0.624 2 high 31 f 1 y r y pre  79 1 0.695 2 high 31 f 1 y r y post 1 0.813 2 high 31 f 1 y r y post 2 0.696 2 high 31 f 1 y r y pre 2 0.485 2 high 31 f 1 y r y pre 2 0.604 2 high 31 f 1 y r y post 2 0.548 2 high 31 f 1 y r y post 3 0.559 2 high 31 f 1 y r y pre  3 0.507 2 high 31 f 1 y r y pre 3 0.596 2 high 31 f 1 y r y post 3 0.702 2 high 31 f 1 y r y post 1 0.533 2 high 31 f 1 n l n pre 1 0.611 2 high 31 f 1 n l n pre 1 0.707 2 high 31 f 1 n l n post 1 0.586 2 high 31 f 1 n l n post 2 0.474 2 high 31 f 1 n l n pre 2 0.407 2 high 31 f 1 n l n pre 2 0.581 2 high 31 f 1 n l n post 2 0.477 2 high 31 f 1 n l n post 3 0.447 2 high 31 f 1 n l n pre  3 0.399 2 high 31 f 1 n l n pre 3 0.581 2 high 31 f 1 n l n post 3 0.521 2 high 31 f 1 n l n post 1 0.494 3 high 31 f 1 y r y pre 1 0.461 3 high 31 f 1 y r y pre 1 0.668 3 high 31 f 1 y r y post 1 0.588 3 high 31 f 1 y r y post 2 0.418 3 high 31 f 1 y r y pre 2 0.471 3 high 31 f 1 y r y pre 2 0.509 3 high 31 f 1 y r y post 2 0.504 3 high 31 f 1 y r y post  80 3 0.396 3 high 31 f 1 y r y pre  3 0.363 3 high 31 f 1 y r y pre 3 0.383 3 high 31 f 1 y r y post 3 0.398 3 high 31 f 1 y r y post 1 0.398 3 high 31 f 1 n l n pre 1 0.328 3 high 31 f 1 n l n pre 1 0.414 3 high 31 f 1 n l n post 1 0.373 3 high 31 f 1 n l n post 2 0.342 3 high 31 f 1 n l n pre 2 0.348 3 high 31 f 1 n l n pre 2 0.31 3 high 31 f 1 n l n post 2 0.376 3 high 31 f 1 n l n post 3 0.371 3 high 31 f 1 n l n pre 3 0.371 3 high 31 f 1 n l n pre 3 0.378 3 high 31 f 1 n l n post 3 0.334 3 high 31 f 1 n l n post 1 0.814 4 low 36 m 1 n r y pre 1 0.838 4 low 36 m 1 n r y pre 1 0.618 4 low 36 m 1 n r y post 1 0.707 4 low 36 m 1 n r y post 2 0.648 4 low 36 m 1 n r y pre 2 0.625 4 low 36 m 1 n r y pre 2 0.699 4 low 36 m 1 n r y post 2 0.725 4 low 36 m 1 n r y post 3 0.678 4 low 36 m 1 n r y pre 3 0.594 4 low 36 m 1 n r y pre 3 0.616 4 low 36 m 1 n r y post 3 0.627 4 low 36 m 1 n r y post 1 0.561 4 low 36 m 1 y l n pre  81 1 0.72 4 low 36 m 1 y l n pre 1 0.506 4 low 36 m 1 y l n post 1 0.606 4 low 36 m 1 y l n post 2 0.583 4 low 36 m 1 y l n pre 2 0.471 4 low 36 m 1 y l n pre 2 0.566 4 low 36 m 1 y l n post 2 0.606 4 low 36 m 1 y l n post 3 0.635 4 low 36 m 1 y l n pre 3 0.652 4 low 36 m 1 y l n pre 3 0.482 4 low 36 m 1 y l n post 3 0.508 4 low 36 m 1 y l n post 1 0.445 5 moderate 34 f 1 y r y pre 1 0.474 5 moderate 34 f 1 y r y pre 1 0.506 5 moderate 34 f 1 y r y post  1 0.667 5 moderate 34 f 1 y r y post  2 0.511 5 moderate 34 f 1 y r y pre 2 0.579 5 moderate 34 f 1 y r y pre 2 0.577 5 moderate 34 f 1 y r y post  2 0.48 5 moderate 34 f 1 y r y post  3 0.468 5 moderate 34 f 1 y r y pre 3 0.555 5 moderate 34 f 1 y r y pre 3 0.613 5 moderate 34 f 1 y r y post  3 0.309 5 moderate 34 f 1 y r y post  1 0.492 5 moderate 34 f 1 n l n pre 1 0.378 5 moderate 34 f 1 n l n pre 1 0.533 5 moderate 34 f 1 n l n post  1 0.443 5 moderate 34 f 1 n l n post  2 0.447 5 moderate 34 f 1 n l n pre 2 0.364 5 moderate 34 f 1 n l n pre  82 2 0.485 5 moderate 34 f 1 n l n post  2 0.509 5 moderate 34 f 1 n l n post  3 0.369 5 moderate 34 f 1 n l n pre 3 0.469 5 moderate 34 f 1 n l n pre 3 0.391 5 moderate 34 f 1 n l n post  3 0.327 5 moderate 34 f 1 n l n post  1 0.889 6 high 35 m 2 y r y pre 1 0.899 6 high 35 m 2 y r y pre 1 0.752 6 high 35 m 2 y r y post  1 0.875 6 high 35 m 2 y r y post  2 0.765 6 high 35 m 2 y r y pre 2 0.822 6 high 35 m 2 y r y pre 2 0.702 6 high 35 m 2 y r y post  2 0.786 6 high 35 m 2 y r y post  3 0.688 6 high 35 m 2 y r y pre 3 0.741 6 high 35 m 2 y r y pre 3 0.682 6 high 35 m 2 y r y post  3 0.689 6 high 35 m 2 y r y post  1 0.832 6 high 35 m 2 n l n pre 1 0.818 6 high 35 m 2 n l n pre 1 0.779 6 high 35 m 2 n l n post  1 0.763 6 high 35 m 2 n l n post  2 0.684 6 high 35 m 2 n l n pre 2 0.863 6 high 35 m 2 n l n pre 2 0.746 6 high 35 m 2 n l n post  2 0.816 6 high 35 m 2 n l n post  3 0.736 6 high 35 m 2 n l n pre 3 0.748 6 high 35 m 2 n l n pre 3 0.697 6 high 35 m 2 n l n post   83 3 0.67 6 high 35 m 2 n l n post  1 0.675 7 high 23 f 2 y r y pre 1 3.842 7 high 23 f 2 y r y pre 2 0.46 7 high 23 f 2 y r y pre 2 0.366 7 high 23 f 2 y r y pre 3 0.399 7 high 23 f 2 y r y pre 3 0.407 7 high 23 f 2 y r y pre 1 0.559 8 high 23 m 2 n r y pre 1 0.602 8 high 23 m 2 n r y pre 1 0.663 8 high 23 m 2 n r y post  1 0.563 8 high 23 m 2 n r y post  2 0.512 8 high 23 m 2 n r y pre 2 0.501 8 high 23 m 2 n r y pre 2 0.584 8 high 23 m 2 n r y post  2 0.634 8 high 23 m 2 n r y post  2 0.508 8 high 23 m 2 n r y pre 3 0.574 8 high 23 m 2 n r y pre 3 0.74 8 high 23 m 2 n r y post  3 0.626 8 high 23 m 2 n r y post  1 0.724 8 high 23 m 2 y l n pre 1 0.861 8 high 23 m 2 y l n pre 1 0.601 8 high 23 m 2 y l n post  1 0.745 8 high 23 m 2 y l n post  2 0.656 8 high 23 m 2 y l n pre 2 0.601 8 high 23 m 2 y l n pre 2 0.551 8 high 23 m 2 y l n post  2 0.669 8 high 23 m 2 y l n post  3 0.61 8 high 23 m 2 y l n pre 3 0.458 8 high 23 m 2 y l n pre  84 3 0.55 8 high 23 m 2 y l n post  3 0.563 8 high 23 m 2 y l n post  1 0.512 9 high 22 m 2 y l n pre 1 0.662 9 high 22 m 2 y l n pre 2 0.569 9 high 22 m 2 y l n pre 2 0.59 9 high 22 m 2 y l n pre 3 0.509 9 high 22 m 2 y l n pre 3 0.571 9 high 22 m 2 y l n pre 4 0.479 9 high 22 m 2 y l n pre 4 0.51 9 high 22 m 2 y l n pre 5 0.482 9 high 22 m 2 y l n pre 5 0.449 9 high 22 m 2 y l n pre 6 0.468 9 high 22 m 2 y l n pre 6 0.436 9 high 22 m 2 y l n pre 1 0.967 10 high 24 m 2 y r y pre 1 0.805 10 high 24 m 2 y r y post  1 0.721 10 high 24 m 2 y r y post  2 0.734 10 high 24 m 2 y r y pre 2 0.646 10 high 24 m 2 y r y post  2 0.782 10 high 24 m 2 y r y post  3 0.601 10 high 24 m 2 y r y pre 3 0.656 10 high 24 m 2 y r y post  3 0.685 10 high 24 m 2 y r y post  1 0.76 10 high 24 m 2 n l y pre 1 0.824 10 high 24 m 2 n l y pre 1 0.909 10 high 24 m 2 n l y post  1 0.842 10 high 24 m 2 n l y post  2 0.763 10 high 24 m 2 n l y pre 2 0.798 10 high 24 m 2 n l y pre  85 2 0.768 10 high 24 m 2 n l y post  2 0.81 10 high 24 m 2 n l y post  3 0.904 10 high 24 m 2 n l y pre 3 0.789 10 high 24 m 2 n l y pre 3 0.719 10 high 24 m 2 n l y post  3 0.723 10 high 24 m 2 n l y post  1 0.43 11 low 37 f 2 n r y pre 1 0.464 11 low 37 f 2 n r y pre 1 0.45 11 low 37 f 2 n r y post  1 0.385 11 low 37 f 2 n r y post  2 0.443 11 low 37 f 2 n r y pre 2 0.392 11 low 37 f 2 n r y pre 2 0.378 11 low 37 f 2 n r y post  2 0.317 11 low 37 f 2 n r y post  3 0.425 11 low 37 f 2 n r y pre 3 0.433 11 low 37 f 2 n r y pre 3 0.403 11 low 37 f 2 n r y post  3 0.415 11 low 37 f 2 n r y post  1 0.415 11 low 37 f 2 y l n pre 1 0.46 11 low 37 f 2 y l n post  1 0.468 11 low 37 f 2 y l n post  2 0.415 11 low 37 f 2 y l n pre 2 0.367 11 low 37 f 2 y l n post  2 0.381 11 low 37 f 2 y l n post  3 0.338 11 low 37 f 2 y l n pre 3 0.334 11 low 37 f 2 y l n post  3 0.315 11 low 37 f 2 y l n post  1 0.522 12 moderate 27 f 2 n r y pre 1 0.44 12 moderate 27 f 2 n r y pre  86 2 0.53 12 moderate 27 f 2 n r y pre 2 0.614 12 moderate 27 f 2 n r y pre 3 0.441 12 moderate 27 f 2 n r y pre 3 0.597 12 moderate 27 f 2 n r y pre 4 0.382 12 moderate 27 f 2 n r y pre 4 0.448 12 moderate 27 f 2 n r y pre 5 0.382 12 moderate 27 f 2 n r y pre 5 0.502 12 moderate 27 f 2 n r y pre 1 0.385 12 moderate 27 f 2 y l n pre 1 0.513 12 moderate 27 f 2 y l n pre 2 0.449 12 moderate 27 f 2 y l n pre 2 0.551 12 moderate 27 f 2 y l n pre 3 0.447 12 moderate 27 f 2 y l n pre 3 0.464 12 moderate 27 f 2 y l n pre 1 0.714 13 high 23 m 2 y r y pre 1 0.606 13 high 23 m 2 y r y pre 1 0.706 13 high 23 m 2 y r y post  1 0.748 13 high 23 m 2 y r y post  2 0.713 13 high 23 m 2 y r y pre 2 0.628 13 high 23 m 2 y r y pre 2 0.637 13 high 23 m 2 y r y post  2 0.594 13 high 23 m 2 y r y post  3 0.569 13 high 23 m 2 y r y pre 3 0.494 13 high 23 m 2 y r y pre 3 0.496 13 high 23 m 2 y r y post  3 0.545 13 high 23 m 2 y r y post  1 0.616 13 high 23 m 2 n l n pre 1 0.596 13 high 23 m 2 n l n pre 1 0.773 13 high 23 m 2 n l n post   87 1 0.701 13 high 23 m 2 n l n post  2 0.561 13 high 23 m 2 n l n pre 2 0.578 13 high 23 m 2 n l n pre 2 0.618 13 high 23 m 2 n l n post  2 0.554 13 high 23 m 2 n l n post  3 0.619 13 high 23 m 2 n l n pre 3 0.527 13 high 23 m 2 n l n pre 3 0.535 13 high 23 m 2 n l n post  3 0.546 13 high 23 m 2 n l n post  1 0.894 14 high 27 m 2 y r y pre 1 0.675 14 high 27 m 2 y r y pre 1 0.792 14 high 27 m 2 y r y post  1 0.667 14 high 27 m 2 y r y post  2 0.833 14 high 27 m 2 y r y pre 2 0.821 14 high 27 m 2 y r y pre 2 0.847 14 high 27 m 2 y r y post  2 0.689 14 high 27 m 2 y r y post  3 0.642 14 high 27 m 2 y r y pre 3 0.846 14 high 27 m 2 y r y pre 3 0.652 14 high 27 m 2 y r y post  3 0.707 14 high 27 m 2 y r y post  1 0.588 14 high 27 m 2 n l n pre 1 0.575 14 high 27 m 2 n l n pre 1 0.706 14 high 27 m 2 n l n post  1 0.559 14 high 27 m 2 n l n post  2 0.612 14 high 27 m 2 n l n pre 2 0.61 14 high 27 m 2 n l n pre 2 0.563 14 high 27 m 2 n l n post  2 0.796 14 high 27 m 2 n l n post   88 3 0.656 14 high 27 m 2 n l n pre 3 0.646 14 high 27 m 2 n l n pre 3 0.702 14 high 27 m 2 n l n post  3 0.608 14 high 27 m 2 n l n post  1 0.636 15 high 30 f 2 n r y pre 1 0.636 15 high 30 f 2 n r y pre 1 0.578 15 high 30 f 2 n r y post  1 0.653 15 high 30 f 2 n r y post  2 0.581 15 high 30 f 2 n r y pre 2 0.574 15 high 30 f 2 n r y pre 2 0.542 15 high 30 f 2 n r y post  2 0.545 15 high 30 f 2 n r y post  3 0.486 15 high 30 f 2 n r y pre 3 0.503 15 high 30 f 2 n r y pre 3 0.369 15 high 30 f 2 n r y post  3 0.489 15 high 30 f 2 n r y post  1 0.658 15 high 30 f 2 y l n pre 1 0.579 15 high 30 f 2 y l n pre 1 0.587 15 high 30 f 2 y l n post  1 0.385 15 high 30 f 2 y l n post  2 0.516 15 high 30 f 2 y l n pre 2 0.511 15 high 30 f 2 y l n pre 2 0.514 15 high 30 f 2 y l n post  2 0.391 15 high 30 f 2 y l n post  3 0.494 15 high 30 f 2 y l n pre 3 0.485 15 high 30 f 2 y l n pre 3 0.39 15 high 30 f 2 y l n post  3 0.358 15 high 30 f 2 y l n post  1 0.595 16 high 27 f 2 n r y pre  89 1 0.495 16 high 27 f 2 n r y pre 1 0.571 16 high 27 f 2 n r y post  1 0.766 16 high 27 f 2 n r y post  2 0.534 16 high 27 f 2 n r y pre 2 0.427 16 high 27 f 2 n r y pre 2 0.511 16 high 27 f 2 n r y post  2 0.659 16 high 27 f 2 n r y post  3 0.592 16 high 27 f 2 n r y pre 3 0.45 16 high 27 f 2 n r y pre 3 0.57 16 high 27 f 2 n r y post  3 0.586 16 high 27 f 2 n r y post  1 0.511 16 high 27 f 2 y l n pre 1 0.506 16 high 27 f 2 y l n post  1 0.521 16 high 27 f 2 y l n post  2 0.417 16 high 27 f 2 y l n pre 2 0.501 16 high 27 f 2 y l n post  2 0.515 16 high 27 f 2 y l n post  3 0.478 16 high 27 f 2 y l n pre 3 0.478 16 high 27 f 2 y l n post  3 0.46 16 high 27 f 2 y l n post  1 0.44 17 low 35 f 1 y r n pre 1 0.471 17 low 35 f 1 y r n pre 2 0.414 17 low 35 f 1 y r n pre 2 0.456 17 low 35 f 1 y r n pre 3 0.419 17 low 35 f 1 y r n pre 3 0.407 17 low 35 f 1 y r n pre 1 0.457 17 low 35 f 1 n l y pre 1 0.423 17 low 35 f 1 n l y pre 2 0.385 17 low 35 f 1 n l y pre  90 2 0.331 17 low 35 f 1 n l y pre 3 0.384 17 low 35 f 1 n l y pre 3 0.355 17 low 35 f 1 n l y pre 1 0.698 18 high 21 f 1 n r y pre 1 0.621 18 high 21 f 1 n r y pre 1 0.732 18 high 21 f 1 n r y post 1 0.517 18 high 21 f 1 n r y post 2 0.63 18 high 21 f 1 n r y pre 2 0.61 18 high 21 f 1 n r y pre 2 0.638 18 high 21 f 1 n r y post 2 0.582 18 high 21 f 1 n r y post 3 0.532 18 high 21 f 1 n r y pre 3 0.516 18 high 21 f 1 n r y pre 3 0.616 18 high 21 f 1 n r y post 3 0.595 18 high 21 f 1 n r y post 1 0.578 18 high 21 f 1 y l n pre 1 0.506 18 high 21 f 1 y l n pre 1 0.586 18 high 21 f 1 y l n post 1 0.424 18 high 21 f 1 y l n post 2 0.58 18 high 21 f 1 y l n pre 2 0.494 18 high 21 f 1 y l n pre 2 0.606 18 high 21 f 1 y l n post 2 0.562 18 high 21 f 1 y l n post 3 0.496 18 high 21 f 1 y l n pre 3 0.432 18 high 21 f 1 y l n pre 3 0.467 18 high 21 f 1 y l n post 3 0.484 18 high 21 f 1 y l n post  91 Appendix F: Muscle Strength Measurements Participant strength (MVC) Activity level Age Sex Cohort Rest Leg Dominant leg Pre/Post 1 200 high 43 m 1 y r y pre 1 180 high 43 m 1 y r y post 1 200 high 43 m 1 n l n pre 1 180 high 43 m 1 n l n post 2 180 high 31 f 1 y r y pre 2 240 high 31 f 1 y r y post 2 180 high 31 f 1 n l n pre 2 240 high 31 f 1 n l n post 3 70 high 31 f 1 y r y pre 3 130 high 31 f 1 y r y post 3 60 high 31 f 1 n l n pre 3 120 high 31 f 1 n l n post 4 195 low 36 m 1 n r y pre 4 240 low 36 m 1 n r y post 4 185 low 36 m 1 y l n pre 4 225 low 36 m 1 y l n post 5 105 moderate 34 f 1 y r y pre 5 150 moderate 34 f 1 y r y pre 5 105 moderate 34 f 1 n l n post 5 150 moderate 34 f 1 n l n post 6 179 high 35 m 2 y r y pre 6 320 high 35 m 2 y r y pre 6 185 high 35 m 2 n l n post 6 320 high 35 m 2 n l n post 7 200 high 23 f 2 y r y pre  92 7 170 high 23 f 2 y r y post 7 120 high 23 f 2 n l n pre 7 155 high 23 f 2 n l n post 8 160 high 23 m 2 n r y pre 8 270 high 23 m 2 n r y post 8 150 high 23 m 2 y l n pre 8 250 high 23 m 2 y l n post 9 200 high 22 m 2 n r y pre 9 245 high 22 m 2 n r y post 9 185 high 22 m 2 y l n pre 9 225 high 22 m 2 y l n post 10 170 high 24 m 2 y r y pre 10 270 high 24 m 2 y r y post 10 170 high 24 m 2 n l n pre 10 270 high 24 m 2 n l n pre 11 100 low 37 f 2 n r y post 11 160 low 37 f 2 n r y post 11 100 low 37 f 2 y l n pre 11 170 low 37 f 2 y l n pre 12 200 moderate 27 f 2 n r y post 12 235 moderate 27 f 2 n r y post 12 200 moderate 27 f 2 y l n pre 12 240 moderate 27 f 2 y l n post 13 210 high 23 m 2 y r y pre 13 270 high 23 m 2 y r y post 13 180 high 23 m 2 n l n pre 13 260 high 23 m 2 n l n post 14 160 high 27 m 2 y r y pre 14 260 high 27 m 2 y r y post  93 14 160 high 27 m 2 n l n pre 14 243 high 27 m 2 n l n post 15 128 high 30 f 2 n r y pre 15 270 high 30 f 2 n r y post 15 130 high 30 f 2 y r y pre 15 260 high 30 f 2 y r y post 16 130 high 27 f 2 n r y pre 16 210 high 27 f 2 n r y pre 16 125 high 27 f 2 y l n post 16 200 high 27 f 2 y l n post 17 100 low 35 f 1 y r n pre 17 160 low 35 f 1 y r n pre 17 100 low 35 f 1 n l n post 17 145 low 35 f 1 n l n post 18 130 high 21 f 1 n r y pre 18 225 high 21 f 1 n r y pre 18 80 high 21 f 1 y l n post 18 220 high 21 f 1 y l n post  94 Appendix G: Training Load Measurements Participant training load (% MVC) Activity level Age Sex Cohort Rest Leg Dominant leg Pre/Post 1 109.85 high 43 m 1 y r y pre 1 114.87 high 43 m 1 y r y post 1 114.01 high 43 m 1 n l n pre 1 108.19 high 43 m 1 n l n post 2 111.86 high 31 f 1 y r y pre 2 94.12 high 31 f 1 y r y post 2 105.38 high 31 f 1 n l n pre 2 90.62 high 31 f 1 n l n post 3 112.04 high 31 f 1 y r y pre 3 80.32 high 31 f 1 y r y post 3 105.97 high 31 f 1 n l n pre 3 91.68 high 31 f 1 n l n post 4 113.27 low 36 m 1 n r y pre 4 83.22 low 36 m 1 n r y post 4 110.54 low 36 m 1 y l n pre 4 86.53 low 36 m 1 y l n post 5 114.13 moderate 34 f 1 y r y pre 5 79.03 moderate 34 f 1 y r y pre 5 114.01 moderate 34 f 1 n l n post 5 75.05 moderate 34 f 1 n l n post 6 109.85 high 35 m 2 y r y pre 6 91.23 high 35 m 2 y r y pre 6 119.39 high 35 m 2 n l n post 6 90.82 high 35 m 2 n l n post 7 141.30 high 23 f 2 y r y pre  95 7 - high 23 f 2 y r y post 7 108.46 high 23 f 2 n l n pre 7 - high 23 f 2 n l n post 8 93.29 high 23 m 2 n r y pre 8 76.56 high 23 m 2 n r y post 8 88.59 high 23 m 2 y l n pre 8 88.76 high 23 m 2 y l n post 9 94.90 high 22 m 2 n r y pre 9 - high 22 m 2 n r y post 9 94.52 high 22 m 2 y l n pre 9 - high 22 m 2 y l n post 10 86.84 high 24 m 2 y r y pre 10 95.74 high 24 m 2 y r y post 10 87.30 high 24 m 2 n l n pre 10 86.73 high 24 m 2 n l n pre 11 112.96 low 37 f 2 n r y post 11 91.04 low 37 f 2 n r y post 11 120.34 low 37 f 2 y l n pre 11 88.34 low 37 f 2 y l n pre 12 127.29 moderate 27 f 2 n r y post 12 - moderate 27 f 2 n r y post 12 113.74 moderate 27 f 2 y l n pre 12 - moderate 27 f 2 y l n post 13 130.21 high 23 m 2 y r y pre 13 76.96 high 23 m 2 y r y post 13 96.03 high 23 m 2 n l n pre 13 86.97 high 23 m 2 n l n post 14 88.55 high 27 m 2 y r y pre 14 82.82 high 27 m 2 y r y post  96 14 85 high 27 m 2 n l n pre 14 83.01 high 27 m 2 n l n post 15 96.62 high 30 f 2 n r y pre 15 68.2 high 30 f 2 n r y post 15 105.61 high 30 f 2 y r y pre 15 68.78 high 30 f 2 y r y post 16 108.68 high 27 f 2 n r y pre 16 81.71 high 27 f 2 n r y pre 16 111.15 high 27 f 2 y l n post 16 86.62 high 27 f 2 y l n post 17 107.07 low 35 f 1 y r n pre 17 - low 35 f 1 y r n pre 17 108.46 low 35 f 1 n l n post 17 - low 35 f 1 n l n post 18 109.13 high 21 f 1 n r y pre 18 83.98 high 21 f 1 n r y pre 18 105.21 high 21 f 1 y l n post 18 88.50 high 21 f 1 y l n post - Drop out  

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