Crossover fatigue after unilateral submaximal eccentric contractions of the knee flexors involves peripheral alterations and perceived fatigue

Abstract

After a unilateral muscle exercise, the performance of the non-exercised contralateral limb muscle can be also impaired. This crossover fatigue phenomenon is still debated in the literature and very few studies have investigated the influence of eccentric contractions. This study was designed to assess neuromuscular adaptations involved in the crossover fatigue of the non-exercised contralateral hamstrings. Seventeen healthy young men performed a unilateral submaximal eccentric exercise of the knee flexors until a 20% reduction in maximal voluntary isometric contraction (MVIC) torque was attained in the exercised limb (EL). Before, immediately after exercise cessation (POST) and 24 hours later, neuromuscular function, global perceived fatigue and perceived muscle soreness were measured in both the EL and non-exercised limb (NEL). At POST, significant reductions in MVIC were observed in the EL (-28.1%, p < 0.001) and in the NEL (-8.5, p < 0.05). Voluntary activation decreased (-6.0, p < 0.05) in the EL only, while potentiated doublet torque were impaired (Dt100Hz -11.6%, p < 0.001 and Dt10Hz -8.1%, p < 0.05) in both the EL and the NEL. Global perceived fatigue significantly increased at POST (p < 0.001). Interestingly, peripheral alterations and global perceived fatigue may account for the crossover fatigue observed immediately after the exercise in the NEL possibly involving systemic adaptations.

Full text

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Crossover fatigue after unilateral submaximal
eccentric contractions of the knee exors involves
peripheral alterations and perceived fatigue
Jennifer Gioda
Université Côte d’Azur, LAMHESS
Florian Monjo
Université Côte d’Azur, LAMHESS
Flavio Da Silva
Université Côte d’Azur, LAMHESS
Baptiste Corcelle
Université Côte d’Azur, LAMHESS
Enzo Piponnier
Université Côte d’Azur, LAMHESS
Jonathan Bredin
Centre de Santé Institut Rossetti-PEP06
Serge S. Colson (  serge.colson@univ-cotedazur.fr )
Université Côte d’Azur, LAMHESS
Article
Keywords: contralateral, performance fatigability, voluntary activation, perceived muscle soreness,
electromyography
Posted Date: August 24th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1978469/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
After a unilateral muscle exercise, the performance of the non-exercised contralateral limb muscle can be
also impaired. This crossover fatigue phenomenon is still debated in the literature and very few studies
have investigated the inuence of eccentric contractions. This study was designed to assess
neuromuscular adaptations involved in the crossover fatigue of the non-exercised contralateral
hamstrings. Seventeen healthy young men performed a unilateral submaximal eccentric exercise of the
knee exors until a 20% reduction in maximal voluntary isometric contraction (MVIC) torque was attained
in the exercised limb (EL). Before, immediately after exercise cessation (POST) and 24 hours later,
neuromuscular function, global perceived fatigue and perceived muscle soreness were measured in both
the EL and non-exercised limb (NEL). At POST, signicant reductions in MVIC were observed in the EL
(-28.1%, p < 0.001) and in the NEL (-8.5, p < 0.05). Voluntary activation decreased (-6.0, p < 0.05) in the EL
only, while potentiated doublet torque were impaired (Dt100Hz -11.6%, p < 0.001 and Dt10Hz -8.1%, p <
0.05) in both the EL and the NEL. Global perceived fatigue signicantly increased at POST (p < 0.001).
Interestingly, peripheral alterations and global perceived fatigue may account for the crossover fatigue
observed immediately after the exercise in the NEL possibly involving systemic adaptations.
Introduction
After a strenuous unilateral exercise, a decline in muscle force or power production of the exercised
muscle and/or an increase in subjective sensations (e.g., perceived fatigue, perceived muscle soreness)
associated with the task performed is commonly reported. This reduced muscle force production dened
as performance fatigability1 is commonly attributed to interactions between peripheral and central
factors including impairments of contractile function distal to the neuromuscular junction and
adjustments within the central nervous system at the spinal and/or the supraspinal level 2.Interestingly,
during maximal or fatiguing unilateral contractions, involuntary increased electromyographic activity and
force production are observed in the non-exercised contralateral homologous muscle3,4. In addition, after
a fatiguing unilateral exercise, performance fatigability can occur in the non-exercised contralateral
muscle, but this observation is still disputed in the literature5.
Since the beginning of the millennium, numerous studies have sought to investigate the so-called
“crossover” fatigue, dened here as the performance fatigability of a non-exercised contralateral
homologous muscle following unilateral muscle exercise of the ipsilateral muscle 6. Although evidenced
many times4,7−11, some studies failed12,13 to report crossover fatigue. A recent systematic review with
meta-analysis5 concluded that the conicting literature neither supports the existence of crossover
fatigue nor “non-local muscle fatigue” (i.e., fatigue occurring in any non-exercised heterologous muscle).
The moderators that were examined (i.e., study design, age, sex, training status, homologous vs.
heterologous muscles, upper body vs. lower body, type of outcome measure, time of measurement,
fatigue severity) had trivial effects on crossover fatigue. However, this meta-analysis did not take the
inuence of the type of contraction (i.e., dynamic or isometric) into consideration. While most of the

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crossover fatigue studies implemented isometric contractions4,7,10,11,14,15, only four studies have used
eccentric contractions, yet providing conicting results8,9,13,16.
Eccentric contractions are known to generate performance fatigability of the exercised muscles that can
be associated with exercise-induced contractile alterations17–19, delayed muscle soreness and pain8,9, as
well as central drive alterations9,17−19. To date, after a unilateral eccentric exercise, increased 16,
unchanged 13 or reduced force production 8,9 is reported in the non-exercised contralateral homologous
muscle. Due to the variability of the study designs, it is extremely challenging to explain the discrepancies
observed among these studies. Interestingly enough, however, when considering the two studies that
have evidenced crossover fatigue 8,9, an interaction between central factors (i.e., reduced voluntary
activation or muscle activity) and subjective sensations (i.e., increased perceived pain) seemed to
contribute to the performance fatigability observed in the contralateral homologous muscle.
In crossover fatigue studies, the performance fatigability of the contralateral homologous muscle is
generally assumed to encompass complex fatigue-induced central adjustments20,21 possibly leading to
reduced central drive to the non-exercised muscle7,9−11. In addition, peripheral factors involving the
dispersion of metabolites through the cardiovascular system and/or the presence of heat shock proteins
in resting muscles have been assumed to account for the decreased performance of the non-exercised
contralateral muscle5,20 As far as we know, only one study reported an impairment of the resting
electrically evoked twitch associated with reduced performance of the contralateral rst dorsal
interosseous muscle4. Finally, subjective sensations such as pain, effort or increased perception of
fatigue could also contribute to the altered performance of the contralateral homologous muscle 5,8,9,20,
even though such measures were scarcely performed in crossover fatigue studies8. Consequently, even if
the underlying mechanisms accounting for crossover fatigue are still debated, it has recently been
proposed that a complex interaction between peripheral and central factors associated with increased
subjective sensations is likely to occur5. This suggestion is in accordance with some of the results
reported in the two crossover fatigue studies using unilateral eccentric exercise8,9, although these two
studies were not included in the meta-analysis.
To date, the occurrence of crossover fatigue in the knee exors is still unknown. In this context, this study
originally investigated the effect of unilateral submaximal eccentric exercise of the knee exors on the
crossover fatigue of the non-exercised contralateral homologous muscle. Concomitant assessment of
neuromuscular function with measures of perceived fatigue and muscle soreness was intended to
ascertain the inuence of both peripheral and central factors and subjective sensations in crossover
fatigue. Based on recent studies8,9, we hypothesized that the submaximal eccentric fatiguing exercise
would induce crossover fatigue, thus involving a cumulative central activation failure, peripheral
alterations in both the exercised and non-exercised muscles and increases in perceived fatigue and
muscle soreness. We also scrutinized the possible inuence of involuntary increased electromyographic
activity of the contralateral non-exercised muscles during the exercise of the ipsilateral muscle on
crossover fatigue. Finally, due to the well-acknowledged long-lasting effect of eccentric exercises on

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performance fatigability8,9, the performance of both the exercised and contralateral non exercised
muscles were assessed 24 hours after the exercise.
Methods
Participants
Based on a recent study reporting cross-over fatigue after eccentric exercise9, G*Power (version 3.1.9.4;
Kiel University, Kiel, Germany) was used to estimate sample size
a priori
. The sample size determination
led to the participation of ten individuals in a repeated-measure, within-between interaction analyses with
a power set at 0.95. Seventeen healthy young men [mean ± standard deviation (SD); age: 23.8 ± 4.6; body
mass: 77.2 ± 10.2 kg; height: 180.5± 3.9 cm] took part in this study. A sensitivity power analysis (α = 0.05,
power = 0.95) led to a large effect of
f
= 0.47 for the sample size of the participants included in this study.
In order to be part of the study, participants had to be involved in regular recreational sport activities, free
of any medical contraindication to physical activity. Neither could they have experienced musculoskeletal,
neurological, or orthopedic disorder in the lower limbs for at least six months. All volunteers were fully
informed about all risks, discomforts, and benets of the study.
Measurements
Knee exors’ torque
A specic ergometer (Hamtech device, Human Kinematic, Carros, France) allowing reliable and
reproducible measurements in isometric and dynamic conditions22 was used to assess the unilateral
force production of knee exors of both lower limbs. A force transducer (S-beam, LS02-s, Tech Co. Ltd,
Shenzhen, China; capacity: 1000N) was positioned 5 cm above the external malleolus on the Achilles
tendon. A Biopac MP 150 (Biopac Systems, Inc., Goleta, CA., USA) was used to record force production at
a sampling frequency of 1 kHz. Force was converted into torque during oine data processing using
participants’ lever arms (i.e., distance between the lateral tibial condyle and the force transducer). The
ergometer also included a potentiometer (P4500, Novotechnik U.S., Inc., Southborough, MA, USA)
allowing measurements of angular velocity. To stabilize the participants’ pelvis during force production,
two elastic bands were positioned ~5cm above participants’ sacroiliac joint and below the gluteal fold.
Isometric contractions were measured on both the exercised (EL) and non-exercised (NEL) lower limbs in
a standardized position with the hip and the knee exion angles set at 40° and 30°, respectively (0° = full
extension). Dynamic eccentric contractions of the EL started from a position combining 65° of hip exion
and 90° of knee exion to a nal position combining 40° of hip exion and 30° of knee exion.
Knee exors’ surface electromyography
Surface electromyographyof the biceps femoris (BF) muscles of both lower limbs was collected using
pairs of surface electrodes (Ag-AgCl, diameter = 10mm; inter-electrode distance = 20mm; Contrôle-
Graphique, Brie-Comte-Robert, France) placed in accordance with the SENIAM recommendations23. A

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reference electrode was placed on the lateral tibial condyle of the tested lower limb. Indelible ink marks
ensured identical repositioning during the entire experiment. Low-resistance impedance (< 3kΩ) was
obtained by shaving and slightly abrading the skin with emery paper. Electromyographic signals were
recorded at a sampling frequency of 2 kHz using the Biopac MP150 system (Biopac Systems, Inc., Goleta,
CA, USA; bandwidth frequency = 10 – 500Hz, common mode rejection ratio = 110dB, Z Input = 1000MΩ,
gain = 1000).
Knee exors’ electrical stimulation
Percutaneous electrical myostimulation (400V and 1ms duration rectangular pulse) was delivered by an
electrical stimulator (DS7, Digitimer Ltd., Hertfordshire, UK) through self-adhesive rectangular electrodes
(5cm × 9cm - Stimex, Wetzlar, Germany) placed on the participants’ lower limbs. The cathode was
positioned on the hamstrings’ proximal part (i.e., below the gluteal fold) and the anode was located at the
popliteal fossa. Electrodes positions were marked on the skin to guarantee similar placement during the
entire experimental procedure. Maximal twitch force and maximal amplitude of the BF compound muscle
action potential were determined at rest by progressive stimulation intensity increments on both lower
limbs. The stimulation intensity was further increased by 20% (EL: 142.1 ± 24.9mA, NEL: 148.2 ± 31.0) to
warrant adequate assessment of knee exors’ neuromuscular function.
Ratings of perceived fatigue and muscle soreness
The notion of perceived fatigue, which was explained to the participants as “a feeling of diminishing
capacity to cope with physical stressors”24 was assessed using the French translated and validated
version of the Rating-of-Fatigue Scale 25. Ratings of perceived fatigue (RPF) accounting for participants’
general fatigue were scored from 0 to10 (0, not fatigued at all; 10, total fatigue and exhaustion – nothing
left).
Perceived muscle soreness (PMS) was recorded in both lower limbs using a visual analog scale. A steady
25-N pressure was applied with a 0.5cm diameter cylindrical object just above and below surface
electromyographyelectrodes of the BF on both the EL and NEL26. Participants had to score their pain
perception from 0 to10 (0, no pain; 10, worst pain).
Experimental procedure
The participants were required to attend the laboratory in three occasions (Fig. 1): i) a familiarization
session, ii) a testing session including the different measurements realized before (PRE) and immediately
after (POST) the unilateral fatiguing exercise (Session 1), and iii) a testing session performed 24 hours
after the fatiguing exercise (POST24; Session 2).
One week prior to the testing session, participants were acquainted with the equipment, the experimental
procedures, and the neuromuscular function assessment (see details below) on both limbs. During the
familiarization session, a particular focus was put on the angular velocity to be maintained (i.e., 10°.s-1

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provided by real time feedback on a screen placed in front of the participants) during the unilateral
eccentric contractions of the EL. Once fully familiarized with all the procedures on both limbs, the 1
Repetition Maximum eccentric contraction (1RM ECC) of the EL was determined by adding loads until the
participant could no longer control the angular velocity of 10°.s-1.
The two testing sessions (i.e., Session 1 - PRE and Session 2 - POST24) started with the measures of RPF
and PMS scores. A standardized warm-up including the “Extender” and the “Diver” exercises 27 and a
single-leg bridges exercise were performed on both limbs for each exercise (i.e., 2 sets of 6 repetitions),
followed by 10 submaximal isometric contractions of both limbs in the isometric testing position. The
intensity of the submaximal contractions (i.e., in the percentage of perceived maximal force production)
was progressively increased (i.e., ~30%, 4 contractions; ~50%, 3 contractions; ~70%, 2 contractions and
~90%, 1 contraction). Then, neuromuscular function was assessed as follows: i) two maximal voluntary
isometric contractions (MVIC) with a 100-Hz superimposed paired electrical stimulation delivered over the
MVIC plateau, followed by potentiated stimulations elicited at rest at 2 (100Hz paired stimulus), 4 (10Hz
paired stimulus) and 6 s (1Hz single stimulus), and ii) the determination of the 1RM ECC of the EL. The
EL was always tested rst followed by the NEL measures. For the measurements performed immediately
after the unilateral fatiguing exercise (i.e., POST), only one MVIC with superimposed and potentiated
stimulations was assessed on both the EL and NEL, followed by the measure of RPF and PMS scores.
Unilateral fatiguing eccentric exercise
The fatiguing exerciseinvolved repetitive sets of ve unilateral eccentric contractions of the EL knee
exors at 80% of the 1RM ECC measured in PRE. Contractions were performed at an angular velocity of
10°.s-1 (i.e., contraction duration: ~5s). After each contraction, participants were passively lifted into the
starting position by experimenters, providing a 10-s rest period. A 25-s rest interspaced sets of
submaximal eccentric contractions. A MVIC of the EL was performed at the end of each set to evaluate
the level of force decrement. Sets were repeated until a 20% MVIC force decrement was reached on EL.
Data analysis
For PRE, POST and POST24 measurements, the highest MVIC peak torque value produced by both lower
limbs was retained for data analysis. Root mean square (RMS) values of BF surface electromyography
signals were calculated during a 500-ms period over MVIC. BF RMS was normalized to the respective BF
compound muscle action potential (i.e., BF RMS/M) recorded at each time point. Potentiated torques
evoked by electrical paired stimuli at100 Hz (Dt100Hz) and 10 Hz (Dt10Hz)were used as peripheral fatigue
indicators. The ratioDt100Hz-to-Dt10Hz (Dt10Hz/Dt100Hz) was also computed. Along with BF RMS/M, the
maximal voluntary activation level (VA) and the central activation ratio (CAR) of the knee exors were
calculated and served as central fatigue indicators 28,29. VA and CAR were computed according to the
following formula:

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During the unilateral fatiguing eccentric exercise, the number of contractions performed and the amount
of total work calculated using torque-time integral (i.e., area under the torque-time curve) were collected.
The amount of work produced was summed in four consecutive periods that represented 25% of the total
duration of the exercise. The MVICs measured at the end of each set and expressed as a percentage of
the initial MVIC value produced at PRE were linearly interpolated between the nearest values at 25%, 50%
and 75% of the number of sets in order to describe the evolution of the participants’ MVIC throughout the
entire duration of the fatiguing exercise. Similarly, the BF RMS of both the EL and NEL (computed over the
entire 5s duration of the contraction) and expressed as a percentage of the initial value measured at PRE
were linearly interpolated between the nearest values at 25%, 50% and 75% of the exercise. The MVIC and
BF RMS values retained at 100% of the number of sets performed corresponded to the value measured
after the last set of each participant.
Statistical analyses
The normality of the distribution of each variable was tested using the Kolmogorov–Smirnov test.For
normally distributed data, a repeated measures two-way ANOVA (limb × time) was performed to assess
fatigue-induced adaptations. For non-normal distribution, the data was processed through the
nonparametricAligned Rank Transform (ART) procedure30 using ARTool (v. 2.1.2) for main, interaction
and post hoc pairwise comparisons (ART-C procedure)31. Separate ANOVAs were performed on ART
responses. The effect size of each ANOVA was estimated from partial eta square (η²p) values and
considered as small when ~0.01, medium when ~0.06 and large when ≥ 0.14. Bonferroni post-hoc tests
were used when a signicant interaction or main effect was observed. The nonparametric Friedman test
with Conover’s post hoc comparison with a Bonferroni correction was conducted on the RPF and PMS
scores of both the EL and NEL. The effect size was calculated from the Kendall’s coecient of
concordance (Kendall’s W). During the unilateral fatiguing eccentric exercise, separate repeated-measures
ANOVAs tested the time course of MVICs and total work and a repeated measures two-way ANOVA (limb
× time) was performed to assess the time course of BF RMS values of both the EL and NEL. Signicance
was set at p < 0.05. Statistical analyses were performed using Statistica (Statsoft, version 8.0 Tulsa, OK,
USA) or JAPS (v. 0.14) software. Unless specied, normally distributed data is expressed as mean ± SD
(standard deviation) and non-normally distributed data is displayed as median (interquartile range) in the
manuscript, in the tables and gures.
Results
Unilateral eccentric fatiguing exercise

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The number of contractions performed and the total work produced were 37.65 ± 14.59 and 54208.33 ±
21034.52 N.m.s-1, respectively.
Throughout the fatiguing exercise, a signicant reduction of both the MVIC (F(4,64)= 43.58; p<0.001;η²p
=0.73) and the torque-time integral (F(3,48) = 4.93; p<0.01; η²p = 0.24) was observed. At the end of the
fatiguing exercise, the MVIC, expressed as a percentage of the initial value, was lower than the MVICs
produced at PRE, 25, 50 and 75% of the fatiguing exercise duration (p<0.001; Fig. 2a). The amount of
work achieved during the last quarter of the fatiguing exercise duration was smaller than the one
produced during the rst quarter (p<0.01; Fig. 2a). A signicant main effect of the limb was noted for the
BF RMS values during the fatiguing exercise (F(1,32) = 71.86; p<0.001; η²p = 0.69; Fig. 2b) with pooled
mean values of the EL (54.35 ± 21.67%) greater than those of the NEL (9.33 ± 4.74%).
PRE, POST and POST24 measurements
MVICs
A signicant limb × time interaction was observed for MVIC (F(2,64)= 9.12; p<0.001; η²p = 0.22). A
signicant reduction in MVIC was observed between PRE and POST in the EL (-28.09 ± 6.47%; p<0.001;
Fig. 3a) and in the NEL (-8.52 ± 16.16%; p<0.05; Fig. 3a). MVIC torques measured at POST24 were no
longer different from PRE values for both limbs (p>0.05).
Central andperipheralfatigue indicators
After ART-C procedure, a signicant limb × time interaction was observed forboth VA (F(5,96) = 11.09;
p<0.001; η²p = 0.37) and CAR values (F(5,96) = 11.41; p<0.001; η²p = 0.37). A signicant decrease in VA
(-6.00 ± 8.24%; p<0.05; Fig. 3b) and CAR (-1.99 ± 2.48%; p<0.01; Table 1) was observed between PRE and
POST in the EL, whereas no signicant modication was noticed in the NEL. At POST24, VA and CAR had
returned to PRE values. Following ART-C procedure, no signicant change was found in the BF RMS/M
values in both limbs (p>0.05; Table 1). After ART procedure, no modication of the BF compound muscle
action potential values was observed (p>0.05; Table 1).
While no limb × time interaction was noted, a signicant time effect was observed inboth
theDt100Hz(F(2,64)= 8.74; p<0.001, η²p = 0.21; Fig. 3c) and the Dt10Hz (F(2,64) = 3.62; p<0.01, η²p = 0.10;
Fig. 3d). Independently of the limb, Dt100Hzand Dt10Hz, decreased between PRE and POST (-11.56 ±
17.94%; p<0.001 and -8.07 ± 24.23%; p<0.05, respectively). POST24 values were not different from PRE
values anymore (p>0.05). No interaction nor main effect was found for the Dt100Hz-to-Dt10Hz ratio
(p>0.05).
Perceived fatigueand muscle soreness scores
RPF scores were signicantly increased at POST compared to PRE (χ² (2) = 26.79; p<0.001; Kendall’s W:
0.42;Table 1). A signicant increase ofPMS scores(χ² (2) = 6.78; p=0.03; Kendall’s W: 0.68;Table 1) was

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noted in the EL with greater scores at POST24 compared to PRE (p<0.05) whereas no modication of the
PMS scores in the NEL was observed (p>0.05).
Table 1. Median (interquartile range) of neuromuscular function and perceived muscle soreness (PMS) of
both the EL (exercised limb) and the NEL (non-exercised limb) as well as global perceived fatigue (RPF).
Bold values are signicant. *, ** and *** signicantly different from PRE values at p<0.05, p<0.01 and
p<0.001, respectively.
BF
Biceps Femoris,
BF RMS/M
Root Mean Square values of BF muscle normalized
to the compound muscle action potential.
PRE POST POST24
Voluntary Activation (%) EL 99.75 (0.19) 97.87 (5.14)*99.74 (0.66)
NEL 99.59 (0.44) 99.60 (0.14) 99.19 (0.61)
Central Activation Ratio (%) EL 99.90 (0.05)  99.09 (1.66)** 99.93 (0.31)
NEL 99.88 (0.16) 99.89 (0.06) 99.76 (0.20)
BF RMS/M (a.u.) EL 0.15 (0.16) 0.12 (0.13) 0.17 (0.08)
NEL 0.07 (0.08) 0.08 (0.07) 0.08 (0.06)
BF compound muscle action potential (mV) EL 3.04 (4.08) 2.33 (5.27) 3.00 (4.61)
NEL 3.04 (2.53) 2.88 (1.78) 2.96 (2.80)
PMS (0-10) EL 0.00 (0.75) 0.25 (1.50)  0.75 (1.50)*
NEL 0.00 (0.50) 0.00 (0.75) 0.25 (1.00)
RPF (0-10) 0.00 (2.00)   7.00 (1.00)*** 2.00 (2.00)
Discussion
This is the rst study that highlights the occurrence of crossover fatigue on the knee exors. The main
ndings of this study were: i) MVICs of both the EL (-28%) and the contralateral NEL (-9%) were impaired
immediately after the exercise, ii) MVIC reduction of both limbs appeared to be related to an overall
impairment of contractile function and an increase in global perceived fatigue, iii) performance
fatigability of the EL also involved voluntary activation failure, and iv) during the exercise of the
ipsilateral EL, involuntary electromyographic activity of the contralateral NEL did not exceed 10% of its
maximal electromyographic activity.
To date,no study has investigated the effects of a unilateral exercise on the performance fatigability of
the knee exors of the exercised limb and its inuence on performance fatigability of the contralateral
homologous muscles.The immediate 28% MVIC decrease of the EL observed in the current investigation
after an eccentric exercise is in line with the previous studies performed on other muscle groups showing

Page 10/18
MVIC impairment ranging from 16 to 50%8,9,13. Eccentric exercise is known to impair muscle contractile
function, generally assessed through the measure of electrically evoked torques,immediately and/or
during many days after the exercise9,17,18. Accordingly, we observed an immediate (i.e., between PRE and
POST) reduction of both Dt100Hz (-12%) and Dt10Hz (-8%) in the EL with no modication of the Dt100Hz-to-
Dt10Hz ratio. Since the BF compound muscle action potential values were unaffected by the eccentric
exercise (i.e., sarcolemma excitability unchanged), the reduction of electrically evoked torques was a
possible consequence of excitation-contraction coupling failure including increased intramuscular
metabolites concentrations leading to impaired calcium release and/or decreased calcium
sensitivity32,33. Structural damage to muscle bers34 often assessed through the measure of perceived
muscle soreness could also contribute to MVIC decreases. Contrary to a previous study performed on the
knee exors35, no modication of PMS scores was observed immediately after our unilateral eccentric
exercise. This contradictory observation might be ascribable to the smaller amount of MVIC decrease
observed here in comparison with Paschalis
et al.
’s35 study (i.e., -28% vs -45%). Consequently, the knee
exors performance fatigability of EL is due to an impaired muscle contractile function involving
excitation-contraction coupling failure.
In addition to peripheral alterations, the presence of immediate central alterations in the EL was
underlined by signicant decreases in knee exors’ VA (-6%) and CAR (-2%).Althoughprevious
experiments36,37 report non-signicant changes ranging from 1.6 to 4.5% of the knee exors’ voluntary
activation after ecologically induced fatigue (i.e., simulated soccer match and repeated sprints), this is
the rst study that has underlined signicant central alterations on this muscle group after a unilateral
submaximal eccentric exercise. So far, only one study reported a 7% signicant decrease of voluntary
activation after a unilateral submaximal eccentric exercise but it was performed on the plantar exor
muscles9. In addition, these authors did not observe a central activation ratio values change. Because the
measures of VA and CAR do not allow dissociating spinal from supraspinal changes,central
alterationsmay have occurred at both levels2,38.The possible rise in intramuscular metabolites
concentrationassociated with fatiguing exercises is known toincrease the inhibitory actions from groups
III-IV afferentswhich arehighly activated during eccentric contractions39,40. Groups III and IV afferents
can modulate the descending central driveat both spinal (i.e., H-reex studies)41–43 and supraspinal level
(i.e., corticospinal excitability studies)19,39,43 after eccentric exercises on other muscle groups. However, it
is extremely challenging to ascertain the exact origin of this modulation due the conicting results
reported in these studies. Central alterations were also assessed through the changes in the
electromyographic activity (i.e., RMS/M) of the BF muscle, but no change was observed. However, only
BF muscle activity was measured, which does not represent the entire descending central drive sent to the
hamstrings. Along with central alterations, another interesting and original result of the present study that
can be noted is the signicant augmented RPF scores noted immediately after the exercise. A recent
study also reported similar observations on the knee extensors after a submaximal fatiguing eccentric
exercise18.Then, we can put forward the hypothesis that oursubmaximal eccentric exercise of the knee
exors is likely to lead to aglobal feeling of fatigue. To summarize, we originally observed that

Page 11/18
aunilateral submaximal eccentric exercise led to impaired performancefatigabilityof the knee exorsof
the EL (including peripheral and central alterations) associated with aconcomitant increase in perceived
fatigue, thus accounting for the possibleinteraction between these two attributes44.
The 28% MVIC decrease observed on the EL led to a signicant 9% MVIC drop in the contralateral NEL,
evidencing crossover fatigue in the knee exors for the rst time. Our result agrees with MVIC
impairments of -13.7% and -7%reported after a unilateral eccentric exercise in the contralateral knee
extensors and plantar exors, respectively8,9. The involuntary increases ofelectromyographic activity and
force production reported in the non-exercised contralateral homologous muscle during maximal or
fatiguing unilateral contractions of the ipsilateral muscle3,4 could partly contribute to crossover fatigue.
Although observed in the rst dorsal interosseous muscle, progressive increases of the electromyographic
activity up to 38% of the maximal activity were reported with a crossover effect of ~10%4. Here though,
the BF electromyographic activity of the NEL remained stable throughout the fatiguing exercise and
corresponded to ~9% of its maximal electromyographic activity recorded during MVIC at PRE.
Consequently, this level of activity is highly unlikely to have contributed to the MVIC reduction of the NEL.
Nevertheless, andtothe same extentas in the EL,both Dt100Hz (-12%) and Dt10Hz (-8%) of the NEL were
reduced immediately after the exercise. To the best of our knowledge, impaired electrically evoked torques
in the contralateral homologous muscle after a unilateral exercise were only reported once in the literature
after isometric exercises4. More recently, even if Marathamuthu
et al
.9, observed a crossover fatigue in the
non-exercised muscle group after a submaximal eccentric exercise, the resting twitch did not change.
Muscle groups studied (i.e., plantar exors
vs.
knee exors) and/or methodological (i.e., single resting
twitch
vs.
double potentiated twitch) differences could account for this discrepancy with our
investigation. A rst explanation for the electrically evoked torques alterations of the present study could
be related to the spreading of metabolites from the EL to the NEL via the cardiovascular system. Although
speculative here, such observations were evidenced in lower limb muscles following an intense upper
limb muscles exercise45,46. Second, in an animal study, the expression ofheat shock proteins increased
immediately in the homologous contralateral muscle following a fatiguing exercise of the ipsilateral
muscle induced by electrical stimulation47. Thanks to their experimental design, these authors concluded
that both the sympathetic system and the neural system (e.g., through group III and IV afferents
activation) were responsible for the higher expression of heat shock proteins in the contralateral muscle
but also in other remote resting muscles. Although future studies should clearly investigate these
adaptations in human contralateral homologous muscles, it may be assumed that our submaximal
eccentric exercise of the knee exors led to a systemic adaptation involving cardiovascular and neural
systems.
A recent study has also suggested that systemic adaptation could explain eccentric-induced crossover
fatigue9. In this study, greater muscle soreness was reported immediately after the exercise and the
authors argued that pain perception contributed to their signicant 3.5% reduction of voluntary activation
observed on the contralateral muscles. As opposed to that, in our study, the PMS scores, VA and CAR of

Page 12/18
the NEL remained unchanged. However, considering the signicant increases in RPF scores, it cannot be
ruled out that a general feeling of fatigue can contribute to a global systemic adaptation accounting for
the crossover fatigue observed in our investigation. Taken together, the peripheral alterations also
observed in the NEL associated with greater perceived fatigue reinforce the possible inuence of a
systemic adaptation after our submaximal eccentric exercise.
Recent eccentric-induced crossover fatigue studies have shown impairment of force in the contralateral
muscles that can last up to 48 hours8,9. We therefore assessed both EL and NEL 24 hours after the
exercise (POST24), but performance fatigability of the knee exors andperceived fatiguehad been fully
recovered. Then, our observations conict with previous studies showing prolonged crossover fatigue.
Although the muscle groups tested and the fatiguing exercise differed from the present investigation,
both studies reported signicant muscle pain up to 48 hours8 and even 72 hours9 in the contralateral
limbs after their eccentric exercise, which could account for the performance fatigability of the non-
exercised muscle. Here, performance fatigability of the knee exors was not signicantly impaired
anymore at POST24, despite the likely presence of muscle damage indicated by the increase in PMS
scores in the EL only. It might be suggested that this perceived muscle soreness at POST24 could still be
involved in the non-signicant -4.6% reduction of the MVIC of the EL. Future studies should ascertain the
long lasting effects of eccentric exercise on crossover fatigue.
Conclusion
Unilateral submaximal eccentric hamstring exercise undeniably induced immediate crossover fatigue.
The decrease in force production observed in both limbs has a different etiology. On the one hand, the
central activation failure and peripheral alterations (i.e., performance fatigability) associated with global
perceived fatigue (i.e., perceptions of fatigue) can account for the exercise-induced fatigue in the EL. On
the other hand, the crossover fatigue in the NEL may be ascribable to a systemic adaptation involving
peripheral alterations and global perceived fatigue. Originally, we observed impaired electrically evoked
torques in the NEL, but the exact mechanism accounting for this result has still to be further investigated.
Finally, a compensatory mechanism involving an involuntary increase of the electromyographic activity in
the NEL did not contribute to the crossover fatigue.
Declarations
Data availability
The datasets generated during the present study are available from the corresponding author on
reasonable request.
Ethics declarations. Approval was obtained from the Ethics Committee (Protection Committee of People
for Biomedical Research Southeast III, France; Authorization Number 2020-A02811-38), were conducted
in accordance with the latest version of the Declaration of Helsinki (2013).

Page 13/18
Acknowledgements
This work has been partly supported byFrench government, through the UCAJEDIInvestments in the
Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-
01.
Author contributions
J.G., F.M., F.D.S., B.C., E.P., J.B., and S.S.C. conceived and designed the experiments. J.G., F.M., F.D.S., B.C.,
and F.M. conducted the experiments. J.G., F.D.S., B.C., J.B., and S.S.C. analyzed data. J.G., and S.S.C.
wrote the rst draft of the manuscript. All authors contributed to the interpretation of the results, edited,
critically revised and approved the nal version of the manuscript.
Competing interests
The authors declare no potential conict of interest.
Consent to participate.
All participants included in the study signed an informed consent form.
Consent for publication.
Participants signed informed consent regarding the anonymized publication of the data collected during
this study.
Additional information
Correspondence and requests for materials should be addressed to S.S.C.
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Figures

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Figure 1
Overview of the experimental procedure. After a familiarization session, a rst session (Session 1)
assessed the neuromuscular function of both the EL (exercised limb) and the NEL (non-exercised limb),
as well as perceived fatigue (RPF) and soreness (PMS), before (PRE) and immediately after (POST) a
unilateral submaximal fatiguing eccentric exercise of the EL knee exors. A second session performed 24
(POST24) hours after the exercise (Session 2) was similar to PRE testing.
MVIC
Maximal voluntary
isometric contraction,
1RM ECC
1 Repetition Maximum eccentric contraction.
Figure 2

Page 18/18
Measures analyzed during the unilateral submaximal eccentric exercise. MVIC expressed as a percentage
of the initial value (black line) and torque-time integral (dashed bars; mean ± standard error) of the
exercised limb (Panel a). Surface electromyography (Panel b) of the biceps femoris muscle (BF RMS)
expressed as a percentage of the initial value of both the EL (exercised limb black bars) and the NEL
(non-exercised limb; gray bars). £££ MVIC signicantly different from initial, 25%, 50% and 75% values at
p<0.001. §§ Torque-time integral produced during the fourth quarter of exercise duration signicantly
different from the rst quarter at p<0.01. *** Pooled BF RMS values of the EL signicantly different from
the NEL at p<0.001.
MVIC
Maximal voluntary isometric contraction.
Figure 3
Effects of the unilateral submaximal eccentric exercise on knee exor’s neuromuscular function of both
the EL (exercised limb) and the NEL (non-exercised limb). MVIC (Panel a), voluntary activation (Panel b),
Dt100Hz (Panel c) and Dt10Hz (Panel d) measured before (PRE; black bars), immediately after (POST; gray
bars) and 24 (POST24; white bars) hours after the exercise. * and *** signicantly different between PRE
and POST at p<0.05 and p<0.001, respectively. $ and $$$ pooled values of both the EL and the NEL
measured at POST signicantly different from pooled EL and NEL values measured at PRE at p<0.05 and
p<0.001, respectively.
MVIC
Maximal voluntary isometric contraction,
Dt100Hz
Potentiated torque at 100
Hz,
Dt10Hz
Potentiated torque at 10 Hz. In this gure, voluntary activation values are displayed as mean ±
standard error, although data was non-normally distributed.