Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.     

The effect of temperature and stimulation scheme on fatigue and recovery in Xenopus muscle fibres

The influence of temperature and alternations of the stimulation scheme on fatigue development and recovery has been studied in single toe muscle fibres of Xenopus. Fatigue was in all cases produced by intermittent tetanic stimulation. In the temperature experiments easily fatigued (type 1) and fatigue-resistant (type 2) fibres were fatigued in successive series at 10.0, 15.0 and 22.5 degrees C. Lowering the temperature did not markedly influence the time-course of fatigue development in either of the fibre types. At 22.5 degrees C these fibres usually display post-contractile depression (PCD), a delayed force suppression, during the recovery period. At the lower temperatures PCD was not observed in type 1 fibres and it was delayed in type 2 fibres. Only type 1 fibres were studied in the altered stimulation scheme experiments. Neither the time-course of fatigue development nor the recovery process was markedly influenced by an alteration of tetanic stimulus frequency in the range of 40-80 Hz. Increasing the time-tension area produced before the standard fatigue level (40% of the original force) was reached, by increasing the initial interval between tetani, caused a more pronounced PCD. From these results it can be concluded that fatigue development and recovery are complex processes which cannot be readily explained by a single mechanism.     

Mitochondrial function in intact skeletal muscle fibres of creatine kinase deficient mice

Creatine kinase (CK) has a central role in skeletal muscle, acting as a fast energy buffer and shuttle between sites of energy production (mitochondria) and consumption (cross-bridges and ion pumps). Unexpectedly, isolated fast-twitch skeletal muscle cells of mice deficient in both cytosolic and mitochondrial CK (CK-/-) are highly fatigue resistant during stimulation protocols that stress aerobic metabolism. We have now studied different aspects of mitochondrial function in CK-/- skeletal muscle. Intact, single fibres of flexor digitorum brevis (FDB) muscles were fatigued by repeated tetanic stimulation (70 Hz, 350 ms duration, duty cycle 0.14). Under control conditions, CK-/- FDB fibres were more fatigue resistant than wild-type fibres. However, after mitochondrial inhibition with cyanide, force declined markedly faster in CK-/- fibres than in wild-type fibres. The rapid force decline in CK-/- fibres was not due to decreased myoplasmic [Ca²⁺] during tetani (measured with indo-1), which in these fibres remained virtually constant during fatigue in the presence of cyanide. Intact, single fibres of highly oxidative soleus muscles were fatigued by repeated tetani (50 Hz, 500 ms duration, duty cycle 0.5). All CK-/- soleus fibres tested ( n = 9) produced > 40% force at the end of the fatiguing stimulation period (500 tetani), whereas force fell to < 40% before 500 tetani in two of three wild-type fibres. Mitochondrial [Ca²⁺] (measured with rhod-2 and confocal microscopy) increased during repeated tetanic stimulation in CK-/- but not in wild-type FDB fibres. In conclusion, mitochondria and energy shuttling operate effectively in CK-/- fibres and this is associated with an increase in mitochondrial [Ca²⁺].     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Moderate fatigue studied at great sarcomere lengths in frog single muscle fibres

Single fibres from the anterior tibialis muscle of Rana temporaria (temperature, 2–3 ^oC) were moderately fatigued at a prestretched sarcomere length (approximately 3.6 μm) by reducing the intervals between 2 s tetani from 240 to 20 s. Changes in length of marked segments along the muscle fibre were monitored during fixed–end tetani using a photoelectric recording system. In contrast to the situation at 2.2 μm sarcomere length, the central region of the fibre was elongated during tetanus at the prestretched fibre length, whereas the segments next to the fibre–tendon junctions (end segments) shortened correspondingly. These length changes were associated with a slow climb of force (tension creep). During development of fatigue, the maximum tetanic tension was reduced to about 78% of the control value and the time course of the tetanus was markedly changed. The tension creep observed in control tetanus was greatly reduced during fatigue, the tetanic tension remaining nearly constant after an initial rising phase. The change in shape of the force myogram during fatigue was associated with a considerable reduction in the amplitude of segment movements along the fibre. The results are explainable by assuming that the end segments (having shorter sarcomeres) become more fatigued than other parts in series along the fibre. At the same time the central segments are likely to have acquired a higher ability to resist stretch by the end segments.     

The effects of temperature on the mechanical performance in fatigued single muscle fibers of the frog induced by twitch and tetanus

Muscle fatigue induced by consecutive twitches or tetani was studied in single skeletal muscle fibers of the frog, Rana japonica. The fatigue by twitch appeared sooner after the start of stimulation at lower temperatures (2-5 degrees C) than at higher ones (15-20 degrees C), while the fatigue by tetanus appeared sooner at higher temperatures. When a twitch-fatigued fiber was bathed in a solution with caffeine (15 mM), the contracture force was much higher than the fatigued force, while in tetanus fatigue, the force by caffeine was not different from the fatigued force. The length-force relation in fatigued fibers was compared with that in pre-fatigue at low and high temperatures. It was noticed that the ascending limb of the length-force curve in fatigued fibers by twitch was lower than that in pre-fatigue at the low temperatures; namely, the fatigue by twitch was more marked in shorter muscle length, while no marked change in the length-force relation was detected in the tetanus fatigue at the low and high temperatures. The maximum shortening velocity, measured by the slack test, decreased in both types of fatigue. These results suggest that the fatigue by twitch may be mainly due to the failure of activation of the contractile system, while in the fatigue by tetanus, the rate of the interaction between actin and myosin may be impaired due to the change in intracellular chemical environment.     

Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres

The firefly luciferin/luciferase reaction was utilized to monitor intracellular ATP concentration ([ATP](i)). Single fibres of mouse skeletal muscle were dissected and injected with luciferase. Luciferin was added to the perfusate and light emission from the fibres was monitored as an indication of [ATP](i). Inhibition of oxidative phosphorylation with cyanide and anaerobic glycolysis with iodoacetate caused light emission to fall to zero within 10 min and the fibres developed a rigor contraction. Inhibition of creatine kinase with 2,4-dinitro-1-fluorobenzene produced a small transient fall in light emission in association with each tetanus. Muscle fibres were fatigued by repeated tetani and 5/12 fibres showed a fall in light emission in the late phase of fatigue. If fibres were allowed to recover from fatigue in the absence of glucose and then restimulated in the absence of glucose they fatigued much more rapidly. However, such fibres showed no obvious change in light emission. We conclude that the luciferin/luciferase system can be used to monitor [ATP](i) in functioning single skeletal muscle cells. A depletion of global [ATP](i) is not observed in all fatiguing fibres and cannot be the sole cause of the final phase of fatigue.     

Recovery of fatiguedXenopus muscle fibres is markedly affected by the extracellular tonicity

Summary In previous studies it has shown that isolatedXenopus muscle fibres may enter a long-lasting, reversible state of severely depressed tetanic force when recovering from fatigue produced by repeated tetani. The mechanism behind this postcontractile depression (PCD) has been studied further by exposing rested and fatigued fibres to a hypertonic (1.2 x normal tonicity) or a hypotonic (0.8x) solution. In the rested state the average tetanic tension increased by 9% in the hypotonic solution and was reduced by 8% in the hypertonic solution. After fatiguing stimulation similar alterations of tonicity resulted in changes of tetanic tension of about 40% in easily fatigable fibres (type 1;n=21); an increased tonicity always resulted in reduced tension, whereas decreased tonicity gave an increased tension output. Similar results were obtained in fatigue-resistant fibres (type 2;n=4), but here the force depression caused by hypertonicity appeared to be irreversible. Thus, fibres were markedly more sensitive to changes of the extracellular tonicity during the recovery period. It is suggested that this increased sensitivity reflects alterations in the signal transmission between t-tubules and sarcoplasmic reticulum.     

The elementary force generation process probed by temperature and length perturbations in muscle fibres from the rabbit

Single chemically permeabilized fibres from rabbit psoas muscle were activated maximally at 5–6 °C and then exposed to a rapid temperature increase ('T-jump') up to 37 °C by passing a high-voltage pulse (40 kHz AC, 0.15 ms duration) through the fibre length. Fibre cooling after the T-jump was compensated by applying a warming (40 kHz AC, 200 ms) pulse. Tension and changes in sarcomere length induced by the T-jumps and by fast length step perturbations of the fibres were monitored. In some experiments sarcomere length feedback control was used. After T-jumps tension increased from ∼55 kN m⁻² at 5–6 °C to ∼270 kN m⁻² at 36–37 °C, while stiffness rose by ∼15 %, suggesting that at a higher temperature the myosin head generates more force. The temperature-tension relation became less steep at temperatures above 25°C, but was not saturated even at near-physiological temperature. Comparison of tension transients induced by the T-jump and length steps showed that they are different. The T-jump transients were several times slower than fast partial tension recovery following length steps at low and high temperature (phase 2). The kinetics of the tension rise after the T-jumps was independent of the preceding length changes. When the length steps were applied during the tension rise induced by the T-jump, the observed complex tension transient was simply the sum of two separate responses to the mechanical and temperature perturbations. This demonstrates the absence of interaction between these processes. The data suggest that tension transients induced by the T-jumps and length steps are caused by different processes in myosin cross-bridges.     

Physiological characteristics of re-innervation of skeletal muscle in the mouse

1. Re-innervation of soleus was studied in the mouse after either crushing the sciatic nerve or re-implanting the nerve to soleus outside the original end-plate region.2. During the early stages of re-innervation subthreshold end-plate potentials (e.p.p.s) were recorded in muscle fibres in response to nerve stimulation. Later the e.p.p.s became large enough to evoke action potentials in muscle fibres.3. The rate of recovery of the release of acetylcholine (ACh) as estimated from the quantal content of e.p.p.s was faster when nerves re-innervated the old end-plate region after nerve crush than after re-implantation so that new neuromuscular junctions were formed.4. During re-innervation soleus fatigued more rapidly than normal during repetitive nerve stimulation. The fatigue was due to failure of neuromuscular transmission associated with an impaired release of ACh.5. During re-innervation soleus was supersensitive to ACh until nerve stimulation was capable of evoking action potentials in muscle fibres.     

Time to fatigue is increased in mouse muscle at 37°C; the role of iron and reactive oxygen species

Studies exploring the rate of fatigue in isolated muscle at 37°C have produced mixed results. In the present study, muscle fibre bundles from the mouse foot were used to study the effect of temperature on the rate of muscle fatigue. Provided iron was excluded from the solutions, time to fatigue at 37°C was increased compared to 22°C (125 ± 8% of 22°C fatigue time). In contrast, when iron was present (∼1 μ m ), fatigue was accelerated (68 ± 10%). Iron can increase reactive oxygen species (ROS), which are believed to accelerate fatigue. The addition of 25–100 μ m H₂O₂ at 22°C reduced time to fatigue to 80–20% of the control, respectively. Iron was added to cultured primary skeletal muscle cells to determine if iron could increase ROS production. Neither iron entry nor ROS production were detected in non-contracting muscle cells. The addition of 8-hydroxyquinoline, which facilitates iron entry, to iron–ascorbic acid solutions caused a rapid rise in intracellular iron and ROS. Our results indicate that time to fatigue in vitro is increased at 37°C relative to 22°C, but the addition of ROS can accelerate fatigue. An increase in muscle iron can accelerate ROS production, which may be important during or following exercise and in haemochromatosis, disuse atrophy and sarcopenia.     

Regulation of myoplasmic Ca(2+) in genetically obese (ob/ob) mouse single skeletal muscle fibres

The present study examined whether calcium handling in skeletal muscle fibres from ob/ob mice was abnormal compared to normal mice. Simultaneous measurements of free myoplasmic calcium and force were made in mouse single intact muscle fibres at rest, during repetitive stimulation and for 30 min afterwards. Fibres were subjected to two bouts of intermittent tetanic contractions 1 h apart. The first bout consisted of 50 tetani only, while during the second bout stimulation was continued until force fell to 40% of control. During a bout of 50 repeated contractions, muscle fibres from ob/ob mice were unable to maintain basal calcium and tetanic calcium transients. During a second series of contractions, muscle fibres from ob/ob mice showed a marked improvement in calcium handling compared to the first series but still fatigued more rapidly than control fibres. It is concluded that calcium handling in skeletal muscle fibres from ob/ob mice is abnormal compared to fibres from normal mice and this contributes to premature fatigue.     

Interpolated twitches in fatiguing single mouse muscle fibres: implications for the assessment of central fatigue

An electrically evoked twitch during a maximal voluntary contraction (twitch interpolation) is frequently used to assess central fatigue. In this study we used intact single muscle fibres to determine if intramuscular mechanisms could affect the force increase with the twitch interpolation technique. Intact single fibres from flexor digitorum brevis of NMRI mice were dissected and mounted in a chamber equipped with a force transducer. Free myoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured with the fluorescent Ca²⁺ indicator indo-1. Seven fibres were fatigued with repeated 70 Hz tetani until 40% initial force with an interpolated pulse evoked every fifth tetanus. Results showed that the force generated by the interpolated twitch increased throughout fatigue, being 9 ± 1% of tetanic force at the start and 19 ± 1% at the end ( P < 0.001). This was not due to a larger increase in [Ca²⁺]_i induced by the interpolated twitch during fatigue but rather to the fact that the force–[Ca²⁺]_i relationship is sigmoidal and fibres entered a steeper part of the relationship during fatigue. In another set of experiments, we observed that repeated tetani evoked at 150 Hz resulted in more rapid fatigue development than at 70 Hz and there was a decrease in force ('sag') during contractions, which was not observed at 70 Hz. In conclusion, the extent of central fatigue is difficult to assess and it may be overestimated when using the twitch interpolation technique.     

Hyperthermia: a failure of the motor cortex and the muscle

Fatigue is increased during hyperthermia, and torque declines more rapidly in sustained maximal voluntary contractions (MVCs). This can be caused by a greater decline in voluntary activation of muscle (i.e. 'central fatigue'). The present study aimed to localize the site of failure of voluntary drive during hyperthermia. Seven subjects made brief (2–3 s) and sustained (2 min) MVCs of elbow flexor muscles in two experiments. Core temperature was normal (∼37°C) in the first experiment, and elevated (∼38.5°C) by passive heating in the second. During some MVCs, transcranial magnetic stimulation of the motor cortex (TMS) was delivered, and the evoked torque (superimposed twitch) and EMG responses were measured. During hyperthermia, voluntary torque was reduced by ∼2.4% during brief MVCs ( P = 0.03), and decreased further (∼12%) during sustained MVCs ( P = 0.01). The superimposed twitch amplitude in the sustained MVC was ∼50% larger ( P = 0.01). Thus, the ability to drive the muscle maximally in a sustained fashion was decreased, and some motor cortical output, which could have increased torque, remained untapped by voluntary drive. The additional central fatigue was not associated with altered motor cortical 'excitability', as EMG responses produced by TMS were similar at the two temperatures. However, the peak relaxation rate of muscle increased by ∼20% ( P = 0.005) during hyperthermia. Hence, faster motor unit firing rates would be required to produce fusion of force. The increased central fatigue during hyperthermia may represent a failure of descending voluntary drive to compensate for changed muscle properties, despite the availability of additional cortical output.     

Fatigue in frog skeletal muscle fibres and effects of methylxanthine derivatives

The influence of theophylline and the related drug caffeine on the mechanical performance of fatigued muscle fibre isolated from semitendinosus muscle of Rana temporaria (2.5–6.7 °C) was investigated. The fibre was stimulated supramaximally to produce 1 s fused tetani and 2 s and 10 s partially fused tetani at intervals of 10 min. Fatigue was produced by shortening the contraction interval to 15 and 30 s. This caused a 15–20% decline in the maximum tension during fused tetanus and a 40–50% decline during partially fused tetanus. Theophylline and caffeine (0.1-0.5 mM) did not change the maximum tension developed by the fatigued fibre during fused tetanization. Both drugs, however, markedly increased the tension output of the fatigued muscle fibre during partially fused tetanus. It was observed that the increase in partially fused tetanic tension by theophylline and caffeine was associated with an increase in the degree of fusion. This later effect was even more pronounced in the presence of diethyl-stilboestrol. It is concluded that these drugs may not reverse the metabolic changes caused by fatigue, since they are unnable to increase fused tetanic force of a fatigued muscle fibre. The increase in partially fused tetanic tension of a fatigued muscle fibre by these drugs is probably due to enhancement of the activator calcium release from the sarcoplasmic reticulum in response to stimulation.     

Metabolic changes associated with the slowing of relaxation in fatigued mouse muscle.

1. The biochemical basis of the slowing of relaxation seen in fatigue has been examined using an isolated mouse soleus preparation. 2. Slowing of relaxation occurred during prolonged tetani under anaerobic conditions when ATP and PC fell and lactate accumulated. 3. Slowing of relaxation was also demonstrated with muscles poisoned with cyanide and iodoacetic acid when there was a fall in ATP and PC but no accumulation of lactate. During a period of anaerobic recovery following a fatiguing tetanus, relaxation became faster at a time when lactate was accumulating in the muscle. 4. It is concluded that the slowing of relaxation in fatigue is not a consequence of lactate accumulation, and a relationship is demonstrated between the ATP content of the muscle and the rate of relaxation in muscles fatigued by prolonged stimulation, 5. Rates of ATP turn-over in fresh muscle, and at intervals throughout a tetanus are consistent with the suggestion that the rate limiting step for myofibrillar ATPase may be directly related to the rate limiting step for the decay of tension during relaxation.     

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo , is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca²⁺ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.     

Force and membrane potential during and after fatiguing, intermittent tetanic stimulation of single Xenopus muscle fibres

Tension and membrane potential have been measured in isolated, short toe muscle fibres of Xenopus during development of fatigue produced by intermittent, tetanic stimulation (0.5 s long 70 Hz trains repeated at 0.3-0.8 Hz) and during recovery. Fibres could be divided into three groups on the basis of their fatigue resistance. In the first group (type 1 fibres) tension fell to 40% of the original after about 70 tetani, in the second group (type 2 fibres) after about 300 tetani and in the third group (type 3 fibres) after about 600 tetani. Recovery was slow in type 1 and 2 fibres and faster in type 3. The former also displayed post-contractile depression (PCD), a 10-40 min period of severely reduced force production, eventually followed by complete recovery. As a result of fatiguing stimulation the membrane potential fell to -70 to -60 mV. It returned to the pre-stimulation value more rapidly than did tension. At the time of maximum PCD most fibres had repolarized to at least -75 mV and action potentials of normal configuration could be elicited. The results of the present experiments underline the importance of defining fibre types in studies of fatigue, also in amphibian muscle. Together with the results of a previous study (L?nnergren & Westerblad 1986) they also demonstrate that both the decline phase and the recovery phase are markedly different when different modes of fatiguing stimulation are used.     

Spatial gradients of intracellular calcium in skeletal muscle during fatigue

We have measured the distribution of intracellular calcium concentration in isolated single muscle fibres fromXenopus laevis using the fluorescent calcium indicator fura-2 with digital imaging fluorescence microscopy. Under control conditions, resting and tetanic calcium were uniform throughout a fibre. When fatigue was produced using a prolonged, high-frequency tetanus, the distribution of calcium within muscle fibres became non-uniform, with greater levels near the outer parts of a fibre than near the centre. This non-uniform distribution of calcium was rapidly abolished by lowering the stimulation frequency. When fatigue was produced using a series of repeated intermittent tetani, tetanic calcium showed an initial small increase, followed by a decrease as stimulation was continued. The distribution of calcium remained uniform under these conditions. Calcium distribution was also uniform during recovery from intermittent tetanic stimulation. Although fibres varied considerably in their fatigue resistance, the time for tension to fall to 50% was correlated with the reduction in tetanic calcium seen at this time. These results indicate that there are at least two patterns of reduced calcium release that can contribute to the development of fatigue. The appearance of a calcium gradient is consistent with impaired t-tubular conduction, while a uniform reduction of calcium is likely to be due to the action of metabolic factors on systems controlling calcium homeostasis within the cell.     

Temperature effect on the rates of isometric force development and relaxation in the fresh and fatigued human adductor pollicis muscle

The purpose of the present study was to investigate the effect of temperature on the rates of isometric force development and relaxation in electrically activated fresh and fatigued human adductor pollicis muscle. Following immersion of the lower arm for 20 min in water baths of four different temperatures, muscle temperatures were approximately 37, 31, 25 and 22 C. Maximal isometric force was reduced by 16.8 +/- 1.5 % at 22 C. The stimulation frequency-force and -rate of force development relationships were shifted to the left at lower temperatures. Q10 values for the maximal rates of force development and relaxation, and the times for 100 to 50 % and 50 to 25 % force relaxation, were about 2.0 between 37 and 25 C and about 3.8 between 25 and 22 C. However, the time for 50 to 25 % force relaxation had a relatively high Q10 value between 25 and 22 C (6.9) and this parameter also appeared to be more sensitive to fatigue compared to the other indices of relaxation. Nevertheless, the effect of fatigue on all parameters decreased with cooling over the entire (37-22 C) temperature range.