Resting shear elastic modulus as a marker of peripheral fatigue during maximal isometric contractions in humans

The aim of this study was to investigate whether the resting Vastus Lateralis (VL) muscle shear elastic modulus (µ), evaluated by shear wave elastography, represents peripheral fatigue during repetition of isometric maximal voluntary contractions (MVCs) of the knee extensor (KE) muscles. Eight healthy well-trained males repeated 60 isometric MVCs of the KE muscles (6 × 10 MVCs; 5 s on/5 s off). Single and double electrical stimulations were delivered to the femoral nerve every ten MVCs during contraction and at rest. The amplitude and properties of the potentiated torque following single (Twpot) double electrostimulation and the amplitude of the concomitant VL compound action potential were considered to be indicators of peripheral fatigue. The resting VLµ was measured during a 5-s rest period after each MVC and electrical stimulation series. The resting VLµ significantly decreased (-21.8 ± 3.9%; P < 0.001) by the end of the fatigue protocol, decreasing from the 10th MVC to the end of the exercise (60th MVC) for all participants, with the loss ranging from 18 to 29%. The potentiated doublet and single twitch torque (Twpot) decreased by 42.5 ± 10.8% and 55.7 ± 8.8%, respectively, by the end of exercise (P < 0.001 for both). The relative mechanical properties of Twpot, i.e. electromechanical delay (P <0 .001), contraction time (P = 0.004), and maximal rate of torque development/relaxation (P < 0.001) also changed significantly during exercise. This study shows that the kinetics of the resting VLµ is associated with changes in both voluntary and electrostimulated torque amplitudes and electromechanical properties of the single twitch during the repetition of maximal voluntary fatiguing exercise. Changes in the resting VLµ may reflect a decline in muscle function, e.g. impairment of excitation-contraction coupling, contractile processes, and/or elastic properties, throughout the increase in muscle compliance, directly affecting force transmission.

values. Absolute KE and KF torque was determined as the peak force reached during maximal 182 efforts. All measurements were taken from the participant's dominant leg (right leg for all 183 participants), which was fixed at 90° (0°= knee fully extended). This muscle length was selected to 184 ensure a good reliability of the neuromuscular assessments. Indeed, it is a length close to the optimal 185 angle of force generating capacity and, at this position the quadriceps muscle is relatively lengthened.

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These last arguments allow us to induce a greater extent of peripheral fatigue and to detect easily 187 muscle stiffness modifications.

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Torque data was corrected for gravity using Cybex software and was acquired and digitized on-line at  values [38]. The superimposed doublet was preferred to a superimposed single T w because it results 198 in a greater signal-to-noise ratio and thus allows the detection of small changes in VAL [39]. The 199 ratio of the amplitude of the Db s100Hz over that of the Db pot100Hz for the relaxed muscle (control to the relaxed muscle) was repeated before the fatigue protocol and every ten MVCs. Peak torque and 206 the Db pot10Hz -to-Db pot100Hz ratio (Db10:100) was then calculated from double pulses; any decrease in 207 this ratio is commonly interpreted as an index of low-frequency fatigue, i.e. the preferential loss of 208 force at low frequencies of electrical stimulation [41]. The following parameters were also obtained 209 from the T wpot response: peak torque (Pt), EMD, CT, HRT, maximal rate of torque development The recording electrodes were taped lengthwise to the skin over the muscle belly, as recommended 217 by SENIAM [42], with an inter-electrode distance of 20 mm. The reference electrode was attached to 218 the patella. Low impedance (Z < 5 k) at the skin-electrode surface was obtained by shaving, gently 219 abrading the skin with thin sand paper, and cleaning with alcohol. EMG signals were amplified (Dual  This RMS value was then normalized to the maximal peak-to-peak amplitude of the potentiated VL   used in shear-wave elastography mode (musculoskeletal preset), as previously described [25].

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Briefly, The SWE technique is based on ultrafast ultrasound sequences that are performed to capture 240 shear-wave propagation. The SWE technique relies on the acoustic radiation force to remotely 241 generate low-frequency shear-waves in tissues, (e.g., muscle, breast, liver), and can be achieved 242 using the same piezoelectric arrays as those used in conventional ultrasonic scanners [25]. The shear- correlates directly with the muscle shear elastic modulus (µ) if the medium is assumed to be purely 248 elastic, which is well accepted in muscle elastography studies [25,26]. The µ was obtained as 249 follows: where p is the muscle density (1,000 kg*m -3 ) and V s is the shear wave speed (in m*s -1 ). This equation 252 implicitly neglects viscous effects. Studies revealed that the shear-wave velocity is almost 253 independent of the frequency of the mechanical shock when measured longitudinally by SWE, 254 demonstrating no significant viscous effects [26].

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An ultrasound probe was placed on the VL muscle at 50% of the distance between the major 256 trochanter and the lateral border of the patella. This position corresponds to the maximal VL ACSA.
it is more sensitive to changes in stiffness than the head of the quadriceps muscle (rectus femoris) 259 after fatiguing from long-duration exercise [22]. Moreover, studies showing decreases in muscle 260 stiffness by assessing aponeurosis or muscle tendon junction displacements after repeated isometric 261 contractions were made exclusively in the VL muscle [2,16] allowing future comparisons with the 262 present study. Shear-wave elastography measurements were carefully standardized by fixing the 263 ultrasound probe using a custom-made system placed over the skin and coating it with a water-  The Aixplorer scanner provided Young's modulus measurements, (E = 3 × p × V s 2 ). The Young's 286 modulus calculation assumes that the material is isotropic, which is clearly not true for muscle [44].

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Thus, all measurements were divided by three to obtain the shear elastic modulus (µ), as in the  between factors, Fisher's LSD post-hoc test was applied to test the discrimination between means.

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The intrasession repeatability of the resting shear elastic modulus was evaluated for the VL muscle 302 between each five measurements, obtained during the 5-s movie, by calculating the intraclass 303 correlation coefficient (ICC) and standard error of measurement (SEM) [45]. Results with a P value < 0.05 were considered to be significant. Statistical procedures were performed using Statistica 8.0 305 software (Statsoft Inc, USA). The results are presented as absolute values (mean ± SD) in Table 1. 306 Data presented in Figs 3-5 are expressed as the percentage of their initial value for the sake of clarity.

Maximal voluntary torque
ANOVA revealed significant absolute and relative (to the control "non-fatigued" value) repetition-dependent effects on MVC torque (P < 0.001, Fig 3A). MVC decreased by 44.4 ± 13.4% by the end of the exercise (P < 0.001), with a significant reduction starting from the 10 th MVC (P = 0.0036).

Peripheral mechanisms of fatigue
Electrically-stimulated potentiated torque ANOVA revealed a significant repetition-dependent effect for both absolute and relative Db pot100Hz and T wpot torque similar to that of MVC (P < 0.001, Fig 3B). Db pot100Hz and Tw pot torque decreased by 42.5 ± 10.8% and 55.7 ± 8.8% by the end of the exercise, respectively (P < 0.001). Reductions in both relative Db pot100Hz and T wpot torque values started from the 10 th MVC (P < 0.001 for both), similarly to MVC.

Electro-Mechanical properties of single twitch torque
ANOVA revealed a significant repetition-dependent effect for both absolute and relative EMD and CT (P < 0.01, Figs 4A and B). The EMD and CT significantly increased during the fatigue protocol, reaching 121.1 ± 6.4% and 120.

Potentiated Vastus Lateralis M-wave amplitude
There were no significant changes in the potentiated VL M max amplitude during the entire fatigue protocol ( Table 1).

VL resting shear elastic modulus
The measurements were reproducible (five measurements obtained during a 5-s clip) throughout

Central mechanisms of fatigue Voluntary activation level
Absolute VAL values are shown in Table 1

EMG Activity
ANOVA revealed there to be no significant effect on the RMS·M max -1 ratio of the VL muscles nor the BF coactivation level over the entire fatigue protocol. Absolute values are presented in Table 1.

Passive EMG activity and torque
Passive KE torque and VL and BF EMG activity assessed during the SWE measurements remained unchanged during the entire fatigue protocol (Table 1).

Discussion
The aim of this study was to investigate whether the resting µ, evaluated by SWE, is a good indicator of peripheral fatigue during the repetition of isometric MVCs of the KE muscles in humans. We hypothesized that a series of isometric MVCs could induce a progressive decline in resting VLµ, reflecting greater muscle compliance and altered muscle function, and that

Reproducibility of measurements
SWE has been demonstrated to be a highly promising alternative to conventional elastography techniques, and provides a reliable and quantitative real-time assessment of muscular tissue stiffness at rest and during isometric contractions or passive stretching [21][22][23]. This study is the first to address the interest of µ on a non-contracted muscle at various stages of the fatigue during a series of maximal isometric contractions of the KE muscles. Methodological factors such as slight probe motion, compression, variability of the measurement, and the ability of subjects to achieve a fully relaxed state need to be meticulously controlled to ensure good reliability of the measurements [46,47]. There was little variability of the measurements (five images obtained during the 5-s clip) during the entire fatigue protocol. ICC and SEM values varied from 96.7% to 99.5% and from 0.20 kPa to 0.26 kPa for the VL muscle, despite the development of fatigue. We achieved this level of reproducibility by positioning a custom-made probe fixation system over the skin and tightly taping it onto the VL muscle (Fig 2B). In addition, the EMG VL and BF EMG activity and passive torque did not change during the SWE recordings (Table 1), verifying that the participants were in a relaxed state.
Our resting VLµ baseline values are concordant with existing published SWE data [29]. The mean and standard deviation of 8.1 ± 2.5 kPa (ranging from 6.0 to 12.3 kPa) for a knee flexion of 90° (0° = knee fully extended) are in accordance with the values of < 10 kPa for the quadriceps muscles for the same knee flexion (90°) in active individuals in a non-fatigued state [29].
However, our control values were higher than those of other studies (~3-5 kPa) for the VL muscle [22,46,48]. These differences may be explained by the short muscle length used in these studies (knee fully extended), as it is known that resting µ and muscle stiffness increase with increasing muscle length [27,29].

Muscle stiffness during and immediately after fatiguing exercise
An understanding of the characteristics of the muscle-tendon complex during and after fatiguing exercise has become essential for the prevention of over-use injuries [49]. Most previous studies have characterized fatigue or training-induced changes in muscle-tendon stiffness by exploring the movement of human tendons and/or aponeurosis structures in vivo [18][19][20]. Several studies have shown greater muscle-tendon compliance (increases in the elongation of connective structures for the same level of the produced force) after repeated contractions of the KE (e.g., VL muscle) and arm muscles [2,16,50]. However, one of the main limitations of these studies is that ultrasound-based techniques reflect modifications in the stiffness of several structures (muscles, tendons, nerves and skin) around a given joint and are not specific to skeletal muscle stiffness.
As mentioned above, SWE provides a quantitative and reliable measurement of individual muscular tissue stiffness [21][22][23]. A strong linear relationship between individual muscle force and dynamic muscle µ, evaluated during contractions, has been demonstrated, suggesting that it may be a good index of individual force [51]. Furthermore, Bouillard et al. [30] confirmed this relationship during submaximal isometric contractions, even when muscle fatigue occurs. These results suggest that SWE can be used to quantify relative modifications in voluntary force, even during fatiguing conditions. In another study [31], the same authors showed that when fatigue was previously induced in one quadriceps muscle (VL), lower dynamic µ values were observed, both initially and during a subsequent submaximal isometric task, relative to the control nonfatigued muscle. Although the amplitude of the muscle µ appears to be affected by fatigue, the underlying mechanisms for its decline are still unknown.
In our study, we observed a progressive decrease in the resting µ of the VL muscle from the 10 th MVC (-9.6 ± 8.5%) to the end of the fatiguing exercise (-21.8 ± 3.9%), suggesting a progressive rise in the VL muscle compliance. Some studies have suggested that increases in muscle-tendon compliance can be explained, in part, by alterations of the viscoelastic properties of the intramuscular connective tissue resulting from the increase in muscle temperature, due to repeated contractions [50,52]. In our study, it is likely that decreases in the muscle µ amplitude were also accompanied by a rise in intramuscular temperature due to repeated maximal isometric contractions. Thus, modifications in the viscoelastic properties of exerted muscles due to fatigue may affect muscle performance. However, the kinetics of muscle temperature during and after the cessation of exercise and its association with the specific viscoelastic properties of muscles is still unknown. Currently, specific viscoelastic properties of the soft tissues may be quantified by realtime supersonic shear imagining, as described in the literature [25,43], but no study has investigated the effects of peripheral fatigue on the viscoelastic properties of skeletal muscle and its role in force transmission.
The decline of the resting µ of the VL muscle found in our study is consistent with those observed for locomotor muscles after strenuous long-distance running, evaluated by invasive techniques, such as tension-myography and muscle belly deformation [24,53], as well as SWE [22]. This exercise-model is clearly very different from that used in our study and the extent of induced fatigue may be greater than that in our model. For example, Andonian et al. [22] observed significant decreases in the resting µ of the quadriceps muscles (without distinguishing between the heads, but mainly in the VL muscle) after an extreme mountain ultra-marathon, which were still reduced after more than 45 hours. However, the relationship between changes in muscle µ and neuromuscular parameters of fatigue was not explored. In contrast to these results, Akagi et al. [54] observed an increase in resting µ (~+7%) of the medial gastrocnemius, but not soleus or lateral gastrocnemius, muscle using another model of fatiguing exercise (1 h at 10% MVC). Lacourpaille et al. [29] showed that intense, non-damaging exercise (3 x 10 concentric MVCs at 120°*s -1 ) did not modify the resting µ of the elbow flexor muscles. They suggested that the resting µ would not be influenced by peripheral factors originating from fatiguing contractions, contrary to those observed in our study. However, in their study, 30 concentric MVCs did not induce a significant decrease in voluntary torque, limiting the conclusions concerning the relationship between fatigue and changes in resting µ. It is possible that in our study the significant decline in resting µ during the fatigue protocol may be closely related to the greater observed extent of exercise-induced peripheral fatigue. To date, there is no consensus among studies concerning modifications of muscle stiffness during and after fatiguing exercise.
Methodological factors, such as the nature of exercise, duration, intensity, muscle length, morphological parameters of the subjects, and technical aspects, such as limb and probe positioning and the subject relaxation state could explain the discrepancies between studies.

Shear elastic modulus and peripheral fatigue outcomes
The mechanisms underlying changes in muscle µ by SWE after fatiguing long-duration exercise are not well described [32]. However, several conclusions have been deduced from modifications in muscle stiffness after damaging exercise. Increases in muscle µ have been mainly observed minutes (30 min) and hours (48 h) after eccentric contractions [27,29]. The authors hypothesized that increases in muscle µ may be strongly associated with the rapid perturbation of calcium homeostasis associated with this exercise modality. However, the relationship between the changes in muscle µ and cellular perturbations was not explored, limiting therefore conclusions.
Moreover, no evaluations were made immediately after exercise in order to determine exerciseinduced fatigue effects as propose in the present study.
We found that significant reductions in the KE MVC were mostly explained by alterations of peripheral rather than central factors (loss of -8.7 ± 8.8% VAL at the 60 th MVC). This is consistent with the literature, showing that high-intensity exercise induces greater peripheral [55] than central fatigue, which is mainly produced by long-duration exercise [40]. with changes in excitation-contraction coupling [1,4,[8][9][10][11], more specifically, impairment of calcium homeostasis, i.e. decreased Ca 2+ release, reuptake, and sensitivity. It has been hypothesized that increases in resting muscle µ following eccentric exercise can be attributed to alterations of calcium homeostasis due to structural disruptions [27,29]. However, we propose that decreases in resting µ in response to fatiguing isometric contractions may be mainly associated with modifications of the elastic properties of the skeletal muscle responsible for force transmission and not by alterations of calcium homeostasis because of the exercise nature differences. The use of SWE at rest appears to be a quantitative and reliable measurement for exploring alterations of peripheral mechanisms during fatiguing contractions. However, the mechanism underlying decreases in resting µ under fatiguing conditions need to be investigated.

Shear elastic modulus and force transmission properties
Single twitch properties, such as the EMD, CT, HRT, and their respective first derivates, the MRTD and MRTR, may also provide information concerning mechanical and electrochemical alterations of skeletal muscle related to force transmission in the fatigued state [12,13]. For example, the EMD refers to the time between the onset of myoelectrical activity from the conduction of the action potentials along the sarcolemma to the development of tension originating from the contractile apparatus and stretching of the series of elastic components (electro-chemical and mechanical processes) [14]. EMD is considered to be a good indicator of the elastic properties of muscle, due to the important role played by elongation of the series of elastic components on total EMD [12,56]. Indeed, it has been demonstrated that the EMD elongate by ~15-20 ms after a fatiguing exercise of KE muscles (25 3-s isometric MVCs), and is associated with a temperature increase [14,57]. These authors suggested that muscle compliance would increase due to muscular fatigue and the subsequent increase of muscle temperature, resulting in a longer time (a higher EMD) to stretch a more elastic muscle. In our study, the EMD significantly increased by 21.1 ± 6.4% (+ 4.5 ms) by the end of the exercise. Moreover, it was related to a significant increase in the CT (20.3 ± 14.5%) and decrease in the MRTD by 56.7 ± 9.0%. The MRTD inversely correlated with overall EMD after fatiguing exercise, in accordance with previous studies [14,58]. Under fatiguing conditions, a lower MRTD would require more time to transfer tension to the tendon insertion point, thus increasing the EMD. In our study, we suggest that alterations on the electromechanical properties of single twitch may have been mainly associated with elastic rather than electrochemical processes (e.g. alteration in the propagation of action potentials along the muscle membrane), due to the lack of modification in the potentiated VL M-wave amplitude during the fatigue protocol. These findings strengthen the relationship between the decline in resting VLµ and alteration of muscle elastic properties (higher muscle compliance) with fatigue. Studies have suggested that fatigue-induced modification of the viscoelastic properties in the muscle-tendon complex could be strongly associated with a longer EMD (mechanical component) [15,59]. These assumptions may explain the higher muscle compliance after fatiguing isometric contractions observed in this study. However, modification of the viscoelastic properties of skeletal muscle due to fatigue is yet to be investigated by supersonic imaging approaches. Thus, in the present study one head of the quadriceps muscle complex was evaluated limiting therefore interpretations about changes of the whole quadriceps neuromuscular properties with fatigue.
Finally, we also found a reduction of 53.8 ± 14.7% at the 60 th MVC in the relaxation phase after muscle contraction (MRTR) with the development of fatigue. It has been suggested that a more compliant elastic component in series requires more time to transmit the decline in cross-bridge tension to the tendon insertion point during relaxation, thus delaying the beginning of force decay [13]. Moreover, it could be related to the decrease in Ca 2+ reuptake by the sarcoplasmic reticulum [60,61]. Surprisingly, we did not observe any modification in the HRT. This suggests that alterations in the elastic properties of the skeletal muscle may have been mainly present during the development of tension originating from the contractile components and stretching of the elastic component in series rather than cross-bridge detachment. However, the high observed inter-individual variability in HRT values may explain this result.

Conclusions
This study shows that the kinetics of resting VLµ may be associated with changes in both voluntary and electrostimulated torque responses and electromechanical properties of the single twitch during the repetition of maximal voluntary fatiguing exercise. Changes in resting VLµ may reflect a decline in muscle function, specifically the elastic properties, by increasing muscle compliance, directly affecting the capacity of force transmission. The use of SWE at rest appears to be a viable alternative and parallel tool to classical neuromuscular methods for the exploration of peripheral fatigue. However, the mechanism underlying the decrease in resting µ under fatiguing conditions still needs to be investigated.
This study provides scientific evidence related to changes of muscle µ associated with greater peripheral fatigue. However, it presents some methodological limitations: (i) the conclusions reported in the present study need be cautiously interpreted due to the small sample (n = 8). A more important sample is required in order to determine whether muscles stiffness is varying as a function of strength loss. Then, (ii) in the present study the whole quadriceps complex was not evaluated limiting interpretation of our results concerning others synergist muscles (RF and VM) and their association with the whole quadriceps neuromuscular assessments. Finally, (iii) it is possible that the magnitude of changes in muscle stiffness was different at other muscle lengths.
Indeed, it has been suggested that muscle stiffness may vary as a function of muscle anatomical configuration (biarticular or monoarticular) [62] and muscle length (short or long) [27]. However, these findings were reported after eccentric but not fatiguing exercise. Thus, the relationship between muscle fatigue, stiffness and muscle length need therefore to be more investigated.
The SWE technique provides a non-invasive, quantitative, and reliable measurement of individual muscle tissue stiffness during fatiguing conditions. The study of both the muscle and tendon characteristics during and after fatiguing exercises in the context of sports medicine or the military is essential for the prevention of over-use injuries resulting from repeated exposure to low or high levels of force. More studies are required to confirm the observed higher specific muscle compliance with fatigue and its direct relationship with modifications of the stiffness of other non-contractile structures by ultrasound. Moreover, the in vivo analysis of the viscoelastic properties of skeletal muscle by supersonic imaging is necessary to better understand modifications in stiffness after fatiguing exercise and its impact on muscle performance.