Short‐latency afferent inhibition is reduced in people with multiple sclerosis during fatiguing muscle contractions

Understanding how inhibitory pathways influence motor cortical activity during fatiguing contractions may provide valuable insight into mechanisms associated with multiple sclerosis (MS) muscle activation. Short‐latency afferent inhibition (SAI) reflects inhibitory interactions between the somatosensory cortex and the motor cortex, and although SAI is typically reduced with MS, it is unknown how SAI is regulated during exercise‐induced fatigue. The current study examined how SAI modulates motor evoked potentials (MEPs) during fatiguing contractions. Fourteen people with relapsing–remitting MS (39 ± 6 years, nine female) and 10 healthy individuals (36 ± 6 years, six female) participated. SAI was induced by stimulation of the median nerve that was paired with TMS over the motor representation of the abductor pollicis brevis. A contraction protocol was employed that depressed force generating capacity using a sustained 3‐min 15% MVC, immediately followed by a low‐intensity (15% MVC) intermittent contraction protocol so that MEP and SAI could be measured during the rest phases of each duty cycle. Similar force, electromyography and MEP responses were observed between groups. However, the MS group had significantly reduced SAI during the contraction protocol compared to the healthy control group (p < .001). Despite the MS group reporting greater scores on the Fatigue Severity Scale and Modified Fatigue Impact Scale, these scales did not correlate with inhibitory measures. As there were no between‐group differences in SSEPs, MS‐related SAI differences during the fatiguing contractions were most likely associated with disease‐related changes in central integration.

K E Y W O R D S motor evoked potential, multiple sclerosis, peripheral nerve stimulation, somatosensory evoked potential, transcranial magnetic stimulation

| INTRODUCTION
Multiple sclerosis (MS) is a progressive, autoimmune, neurodegenerative disease that is characterized by axonal demyelination and damage within the central nervous system (CNS).Given that degeneration of axons can impair neural transmission essential for voluntary muscle activation and motor control (Kent-Braun et al., 1997), MS is typically associated with muscle weakness and fatigue.Indeed, about 80% of individuals report that fatigue is their most debilitating symptom (Kluger et al., 2013;Kobelt et al., 2017).Fatigue that is reported with MS is typically described as a subjective feeling of tiredness, low energy, reduced motivation and difficulty concentrating (Enoka et al., 2021).However, symptoms of fatigue are also associated with motor fatigability, so it is not surprising that people with MS often exhibit greater reductions in muscle force (Brotherton et al., 2022;Coates et al., 2020;Latash et al., 1996;Severijns et al., 2019;Sheean et al., 1997;Steens et al., 2012;Wolkorte et al., 2016) and voluntary muscle activation (Brotherton et al., 2022;Leodori et al., 2023;Sheean et al., 1997;Steens et al., 2012;Wolkorte et al., 2016) compared to healthy individuals performing the same tasks.Interestingly, a number of recent studies show that during both maximal effort (Coates et al., 2020;Latash et al., 1996;Ng et al., 2004;Sheean et al., 1997;Steens et al., 2012;Wolkorte et al., 2016) and low-intensity fatiguing muscle contractions (Brotherton et al., 2022;Latash et al., 1996;Severijns et al., 2019), corticospinal excitability is similar in MS patients and healthy controls.This observation may suggest that intracortical networks in MS patients disinhibit cortical motor circuits to try and maintain descending drive to the muscle during fatiguing contractions.
We recently used transcranial magnetic stimulation (TMS) and motor nerve stimulation to examine muscle activation in people with MS during low-intensity (Brotherton et al., 2022) and maximal elbow flexion contraction tasks (Brotherton et al., 2023).Muscle activation was compromised in people with MS during both contraction tasks compared to healthy controls.However, corticospinal excitability was similar in both tasks between groups, demonstrating that reductions in muscle activation are not necessarily associated with the responsiveness of corticospinal tract.These findings align with recent studies that show similar corticospinal excitability in MS patients and healthy controls during both lowintensity (Brotherton et al., 2022;Latash et al., 1996;Severijns et al., 2019) and maximal fatiguing muscle contractions (Coates et al., 2020;Latash et al., 1996;Ng et al., 2004;Sheean et al., 1997;Steens et al., 2012;Wolkorte et al., 2016).These observations support the theory that intracortical networks in MS patients may be altered to maintain descending drive to the muscle during fatiguing contraction tasks (Filippi et al., 2002;Liepert et al., 2005;Tartaglia et al., 2008).
Short-latency afferent inhibition (SAI) is a phenomenon that reflects inhibitory interactions between the somatosensory cortex and the primary motor cortex (M1) and is mediated by cholinergic and GABAergic inhibitory circuits in the cortex (Di Lazzaro et al., 2000, 2002;Tokimura et al., 2000;Turco et al., 2018).The assessment of SAI in the MS population is particularly relevant given that cholinergic dysfunction in this cohort has been previously documented (Kooi et al., 2011;Ruberg et al., 1987).The modulation of motor evoked potentials (MEP) induced by TMS is the key outcome of SAI, where sensory input to the cortex results in inhibitory postsynaptic potentials in motor neurons (Spampinato et al., 2023;Tokimura et al., 2000;Turco et al., 2018;Turco et al., 2021).Thus, the strength of SAI is influenced by the amplitude of the sensory afferent volley, where greater inhibition is associated with a greater volume of sensory afference (Bailey et al., 2016).Given that a significant aspect of MS pathology is the disruption of afferent pathways, which transmit somatosensory information to the CNS, it is not surprising that SAI is reduced in primary-progressive MS patients (Dubbioso et al., 2022) and relapsing-remitting MS patients (Cucurachi et al., 2008;Mango et al., 2019).However, these studies have consistently examined SAI in people with MS under resting conditions, so it is unknown how SAI is regulated with muscle contractions.Although investigations in healthy individuals have demonstrated that SAI decreases in response to fatiguing contractions of the upper limb (Asmussen et al., 2013;Miyaguchi et al., 2017), the role of SAI in regulating MEPs during fatiguing contractions in people with MS remains unexplored.
The purpose of this study was to examine the modulation of MEPs by SAI during fatiguing contractions in people with MS.SAI was induced by homotopic stimulation of the median nerve that was paired with TMS applied to the motor cortex.A contraction protocol was employed where SAI could be measured from the abductor pollicis brevis (APB) during the rest periods of intermittent, lowintensity, fatiguing contractions.It was hypothesized that (unconditioned) MEP amplitude would be similar between the MS group and a healthy control group during the performance of fatiguing contractions.However, it was also hypothesized that modulation of MEP amplitude by SAI would be reduced in the MS group, which would reflect less intracortical afferent inhibition in people with MS.

| Participants and ethical approval
Fourteen people with relapsing-remitting MS were recruited into the study (39 ± 6 years, nine female).An additional 10 individuals without MS were recruited to act as healthy control participants (36 ± 6 years, six female).All volunteers were right-handed except for one control participant.People with MS were excluded from the study if they had experienced a relapse within 30 days of testing, peripheral nerve pathology, or any neurological comorbidities.All participants completed a medical history questionnaire to ensure that they had no contraindications to TMS and peripheral nerve stimulation or taking any medications that would affect the excitability of motor circuits (such as antidepressants, anticholinergics, and benzodiazepines).Medication schedules for the MS group were maintained during testing.All participants were asked to refrain from any form of CNS stimulant or depressant such as caffeine, alcohol or moderate-to-high intensity exercise for 12 h before testing.Each participant provided written informed consent prior to undertaking testing.All experimental procedures were approved by the Griffith University Human Research Ethics Committee (reference number: 2020/338) and conducted according to the standards set by the Declaration of Helsinki.

| Force and electromyography
Participants sat reclined in a chair with their back and neck supported, and both arms placed in a relaxed position on the arm rests.All measurements were made from the dominant hand, which was placed in a custom force transducer designed to measure isometric abduction force of the thumb (MLP-25 load cell: Transducer Techniques, range 111 N, sensitivity of 2 mV/V).The apparatus positioned the thumb 45 from the midline of the hand, and affixed digits 2 to 5 so that each finger was pointing straight in front of the hand.Surface EMG was obtained from the APB of the dominant hand by using two Ag/AgCl electrodes (Kendall ARBO, 24 mm) in a muscletendon configuration.EMG signals were amplified (Â1000) and band-pass filtered (3-3000 Hz) using a 2nd order Butterworth filter (CED 1902, Cambridge Electronic Design Ltd., UK).All EMG was sampled at 5000 Hz using a Power 1401 data acquisition interface with Spike2 software (version 7.02, Cambridge Electronic Design Ltd., UK).

| Somatosensory evoked potentials
Evoked potentials were elicited in the median nerve of the dominant hand using a constant current stimulator (DS7AH, Digitimer Ltd., UK).After identifying the median nerve with a motorpoint pen electrode, a surface cathode was positioned over the median nerve $3 cm proximal to the wrist and a surface anode was placed over the median nerve $6 cm proximal to the wrist.Single electrical pulses (200 μs) were delivered to the nerve, and responses to stimulation were recorded at the contralateral sensory cortex.An active recording electrode was placed on the scalp 7 cm lateral (C3) and 2 cm posterior from the vertex, and a reference electrode was placed 5 cm anterior to the vertex (Fz).Evoked potentials were sampled at 5000 Hz, amplified Â1000, and band-pass filtered using cutoff frequencies of 3-30 kHz (CED 1902, Cambridge Electronic Design Ltd., UK).The APB resting motor threshold (RMT) was found by starting the stimulator intensity at 20 mA and decreasing the output by 1 mA until an EMG response was no longer visible.Stimulator intensity was set at +5 mA of the RMT when performing SSEP testing (MS: 15.9 ± 6.5 mA; controls: 12.6 ± 2.6 mA, p = .168).A total of 50 SSEPs were averaged to determine the amplitude and latency of the N20 peak.

| MEPs
A MagStim 200 2 TMS unit with a figure-of-eight coil (MagStim Co., UK) was used to generate MEPs in the APB of the dominant hand (Figure 1a).The TMS coil was oriented in an anterior-to-posterior current flow and was positioned $5 cm lateral and $2 cm anterior from the vertex on the contralateral hemisphere to activate the motor cortical representation of the APB.The corresponding site was then clearly marked with reference lines to ensure that the TMS coil was consistently positioned.The RMT was determined by adjusting TMS intensity by 1% increments of the maximal stimulator output (MSO) until an MEP with peak-to-peak amplitude of $50 μV was identified in five out of ten trials.The stimulator intensity that generated a peak-to-peak MEP amplitude of 1 mV was also identified and used for all subsequent data collection involving MEPs (MS: 58.2% ± 10.8% MSO; controls: 54.9% ± 12.7% MSO, p = .446).

| SAI
Conditioning stimuli were applied via electrical stimulation of the median nerve, and test stimuli were applied via TMS of the motor cortex using the equipment and setup outlined above.Conditioning pulses 200 μs in duration were delivered at a constant current stimulator intensity +5 mA of the RMT, and test pulses were delivered at a TMS intensity that generated a peak-to-peak MEP amplitude of 1 mV.A range of interstimulus intervals (ISIs) were used to quantify inhibition, where measuring SAI at multiple ISIs enhanced the ability to examine inhibitory responses in people with MS during our contraction protocol.Thus, the conditioning stimuli proceeded TMS at four different ISIs: N20 latency +2, +4, +6 and + 8 ms (Liepert et al., 2005).Overall, each block of SAI measurements involved five measurements, which consisted of one unconditioned MEP followed by four conditioned MEPs.SAI was always performed when the APB was relaxed and not while the muscle was contracting.

| Testing protocol
Participants attended one testing session.Trait levels of perceived fatigability were quantified prior to testing with the Fatigue Severity Scale (FSS) and the Modified Fatigue Impact Scale (MFIS).SAI, MEP, EMG and force data were collected at baseline, during an exercise-induced fatiguing contraction protocol, and during recovery.

| Baseline measurements
Three brief ($4 s) maximal voluntary contractions (MVC) were performed with the APB muscle to determine the maximal force generating capacity for each individual (Figure 1b).The highest peak torque was used to calculate the submaximal target force of 15% MVC for baseline submaximal contractions and the exerciseinduced fatiguing task.Three brief ($4 s) 15% MVCs were then performed to obtain baseline EMG and MEP responses to TMS (Figure 1b).Motor cortical stimulations were delivered during all maximal and submaximal contractions to evoke MEPs in the APB muscle.SAI measurements were obtained in the resting muscle prior to, F I G U R E 1 Experimental setup and protocol.(a) Transcranial magnetic stimulation was applied to the motor cortex to elicit motor evoked potentials (MEPs) in abductor pollicis brevis (APB).Single unconditioned MEPs provided an index of corticospinal excitability while MEPs conditioned by preceding peripheral nerve stimulation were obtained to measure short-latency afferent inhibition (SAI).Unconditioned and conditioned MEPs were obtained during baseline contractions (b), throughout the contraction protocol (c) and in recovery (d).The fatiguing contraction protocol involved a 3-min sustained 15% maximum voluntary contraction (MVC) of the APB and then concluded with a MVC to quantify the decline in a force relative to baseline measures.Subsequently, 15 duty cycles at 15% MVC commenced where 20 s contractions were paired with 15 s of rest.The intermittent contractions concluded with an MVC.Recovery contractions consisted of a brief 15% MVC followed by a brief MVC at 1, 2, 4 and 6 min, where MEPs and SAIs were again collected.
and following, both contraction types.A rest period of at least 3 and 1 min was given between maximal contractions and submaximal contractions, respectively.

| Fatiguing contraction protocol
Participants performed a 3-min sustained contraction of the APB at 15% MVC, followed by a brief MVC to measure the decline in maximal force generating capacity due to the 3-min contraction (Figure 1c).Our previous work has demonstrated that this protocol significantly reduces MVC force in elbow flexors and is sufficient to reduce the capacity of the CNS to activate muscle (Brotherton et al., 2022).Motor cortical stimulation was delivered every 30 s during the sustained contraction task, and during the brief MVCs, to obtained MEPs during contractions.Immediately following the sustained contraction, a series of intermittent contractions were performed.This intermittent contraction protocol was designed so that a block of SAI measurements could be obtained during periods without muscle activity (the rest phases) while also maintaining the depression in maximal force generating capacity that was induced from the 3-min sustained contraction (the contraction phases).Specifically, participants performed 15 consecutive duty cycles of 20 s contraction at 15% MVC and 15 s rest.Paired stimulations of the motor cortex and peripheral nerve was used to obtain SAI measurements in the relaxed APB during the rest phases, and motor cortical stimulation was delivered during the contraction phases of the duty cycles to obtained MEPs.

| Recovery measurements
Participants performed a sequence of contractions consisting of a brief 15% MVC, followed by a brief MVC 1, 2, 4 and 6 min following the fatiguing contraction protocol (Figure 1d).Similar to the fatiguing contraction protocol, MEPs were measured during rest and during contractions, and SAI was measured when the APB was at rest.

| Data analysis
Somatosensory data were analysed in Signal version 6.05 b (Cambridge Electronic Design).The N20 was calculated as the time taken from the stimulus artefact recorded in the electroencephalography signal to the negative response observed $20 ms after median nerve stimulation.SSEP was then calculated as the peak-to-peak amplitude of N20.Force and EMG data were analysed offline using Spike2 version 7.02 (Cambridge Electronic Design).MVC amplitude was calculated from the peak force generated during the maximal contraction root mean square of the EMG (EMG RMS) for APB was measured from a 200 ms window immediately prior to stimulus artefact for 15% MVC and MVC tasks.TMS-evoked responses were also extracted from the EMG signal.Peak-to-peak MEP amplitude was calculated between vertical cursors that included all phases of the waveform, and the silent period was calculated as the duration from the stimulus artefact to the onset of muscle activity.MEP latency was measured as the duration from the TMS stimulus artefact to the onset of the MEP waveform in the EMG signal.SAI was calculated as the reciprocal of the ratio between conditioned MEPs and unconditioned MEPs (test MEP), where SAI = 1-(mean of conditioned MEPs at all ISIs/mean of test MEP).Thus, a SAI approaching one indicates increasing inhibition, whereas a SAI approaching zero indicates decreasing inhibition.

| Statistical analysis
All analyses were performed in R using RStudio (version 4.3.1;R Foundation for Statistical Computing, Vienna, Austria).Student's t-tests were used to determine differences in participant characteristics and neurophysiological parameters between groups at baseline.Normality of data was assessed by Shapiro-Wilk tests and quantilequantile plots before within and between-group comparisons were made.Mauchly's test was used to identify data that violated sphericity, and when significant, Greenhouse-Geisser corrections were applied.Data was split into phases to separately analyse responses during the sustained contraction, during the duty cycles, and during the recovery phase.Linear mixed effect models were used to examine the main effects of group (MS and control), time (baseline = 0; sustained contraction = 0.5, 1, 1.5, 2, 2.5, 3; duty cycles = 1-15; recovery = 1, 2, 4, 6) on outcome parameters (APB force, APB RMS EMG, APB MEP peak-to-peak amplitude, MEP silent period and short afferent inhibition) using lmerTest package in R. Models were created by iteratively adding predictor variables or interaction effects and determined by model comparison using an ANOVA.The model with the lowest Akaike Information Criterion (AIC) and statistical significance (p < .05)was deemed to model of best fit for that parameter.The model of best fit consisted of group and time as fixed effects, with a random intercept for each subject when accounting for changes in FSS scores (e.g., parameter $group + time + FSS + 1jsubject ID).Interaction effects between group and time, and group and FSS were also examined.Where an interaction effect was detected, Tukey post hoc tests were performed to determine the time points that group differences were identified (emmeans package in R).Bonferroni corrections were applied to the statistical analysis to account for multiple comparisons.Separate linear regressions were performed to determine if fatigue scores (FSS and MFIS) were correlated with participant characteristics and TMS-related inhibitory data.Correlations were performed separately for the two groups (MS and controls) for measurements at baseline, following the sustained fatiguing contraction, following the duty cycles, and in recovery.The significance level was set at .05 for all statistical tests.Data in text is presented as mean and standard deviation.

| Participant characteristics and baseline variables
Both the MS group and the heathy control group has a similar age, weight and height (Table 1).The MS group had an average disease duration of 7 years and reported significantly higher subjective fatigue levels in the MFIS (p < .001)and the FSS ( p < .001).There were no other group differences detected in baseline measurements.

| Voluntary force during the exercise protocol and in recovery
The sustained 15% MVC induced a decline in force that was similar in both groups (main effect of time: F 1,21.7 = 41.876,p < .001;main effect of group: F 1,37.9 = 0.265, p = .610)where MVCs were reduced to 29.0% ± 14.3% for the MS group and 26.9% ± 10.2% for the healthy control group immediately following the 3-min sustained contraction (Figure 2).There was no group Â time interaction detected for force following the sustained contraction (F 1,22.03= 0.130, p = .072).Following the submaximal duty cycles, MVCs for the MS group was 74.6% ± 14.3% of baseline MVC, and MVC for the healthy control group was 75.8% ± 11.1% of baseline MVC (p = .851).At the completion of the duty cycles, the ability to generate force increased (main effect of time: F 1,72.1 = 19.280,p < .001),and after 6 min of recovery, MVC force had returned to $85% of baseline measures for both groups.There was no main effect of group (F 1,13.5 = 0.014, p = .910)or group Â time interaction (F 1,13.6 = 0.035, p = .696)detected during the recovery phase.
F I G U R E 2 Voluntary force during the sustained and intermittent 15% maximal voluntary contraction (MVC) protocol and during recovery.Participants performed a 15% MVC contraction protocol consisting of a sustained 3 min effort, 15 duty cycles and four contraction sequences in recovery.Brief MVCs were performed at baseline, following the sustained contraction, following the duty cycles and during recovery.Force is expressed relative to peak force performed during baseline measures.No between group differences were identified for any phase of the protocol.All data are presented as mean ± SD (MS n = 14, control n = 10).

| EMG and MEPs during brief MVCs throughout the contraction protocol
Brief MVCs were performed at baseline, immediately following the sustained contraction and duty cycles, and intermittently throughout the recovery phase.The RMS EMG during the MVC immediately following the sustained contraction decreased from baseline in both groups (main effect of time: F 1,43 = 10.343,p = .003;Figure 4a), with no group differences (main effect of group: F 1,43 = 2.301, p = .137)and no group Â time interaction detected (F 1,43 = 0.782, p = .382).Following the duty cycles, RMS EMG in the MVC began to increase in both groups to the same extent (main effect of time: F 4, 81.5 = 7.236, p = .009;main effect of group: F 1,21.5 = 2.872, p = .105),and after 6 min in recovery, had returned to baseline levels.There was no group Â time interaction detected for the RMS EMG during the MVCs throughout the contraction protocol (F 4,74.4 = 0.630, p = .643).
When MEPs were conditioned by preceding median nerve stimulation, there were marked changes in the MEP profile for the MS group, which was reflected in SAI calculations.Immediately following the sustained contractions, SAI decreased from baseline in the MS group, whereas the control group demonstrated increased SAI values from baseline.There was no main effect of time (F 1,22.331= 1.049, p = .317)or group (F 1,28.772= 1.744, p = .197),but there was a group Â time interaction detected (F 1,21.997= 7.834, p = .010)where there was a significantly different SAI detected between groups following the sustained contraction ( p = .001).The MS group also consistently demonstrated lower SAI measurements than controls throughout the duty cycles (main effect of group: F 1,45.959 = 15.020,p < .001).However, there was no main effect of time (F 1301.060= 1.200, p = .274)and no group Â time interaction (F 1, 301.968 = 1.850, p = .175)detected throughout the duty cycles.During the recovery phase, SAI measurements remained lower for the MS group than controls (main effect of group: F 1, 45.263 = 15.899,p < .001).There was no main effect of time (F 1, 54.881 = 1.160, p = .286)or a group Â time interaction detected for SAI during recovery (F 1,54.870= 3.559, p = .064).
F I G U R E 4 EMG and responses to motor cortical stimulation during brief maximal voluntary contractions (MVCs) performed at baseline, and during the contraction and recovery phases.RMS EMG amplitude for the abductor pollicis brevis (a), abductor pollicis brevis motor evoked potential (MEP) peak-to-peak amplitude (b), and MEP silent period for the abductor pollicis brevis obtained at baseline, following the sustained contraction, duty cycles and in the recovery phase.RMS EMG, MEP and MEP silent period are expressed relative to measures taken during baseline MVCs.Hash symbol indicates a main effect of time and cross symbols indicates a group Â time interaction (p < .05).All data are presented as mean ± SD (MS group n = 14, control group n = 10).

| Correlation between fatigue scales and neurophysiological measures
FSS and MFIS were significantly correlated for the MS group (r = 0.700, p < .01)but not for the control group (Table 2; r = À0.459,p = .182).For the MS group, a significant positive relationship existed between FSS and the MFIS scores where elevated FSS scores were strongly related to elevated MFIS scores.
In the control group, both FSS (r = À0.652,p = .041)and MFIS (r = 0.793, p = .006)were significantly correlated to the MEP silent period during baseline MVC measurements.FSS was also significantly correlated to baseline SAI (r = À0.648,p = .043),where high FSS scores were associated with a lower SAI.MFIS was significantly correlated with the silent period during the MVCs following the sustained contraction (r = 0.839, p = .002)and in recovery (r = 0.660, F I G U R E 5 Resting unconditioned motor evoked potentials (MEPs) and short-latency afferent inhibition (SAI) measurements taken at baseline, following the 3 min sustained contraction, throughout duty cycles, and in recovery.Unconditioned MEPs (a) evoked by TMS in the resting muscle are expressed as a ratio to MEPs evoked at baseline.Short afferent inhibition values (b) are expressed as a ratio of the conditioned MEP to the unconditioned MEP evoked by TMS at the same time point.A larger SAI indicates more inhibition, with a smaller SAI indicating less inhibition.Asterisk indicates a main effect of group, and cross symbols indicate group Â time interaction (p < .05).All data are presented as mean ± SD (MS group n = 14, control group n = 10).p = .038),but not following the duty cycles (r = 0.628, p = .052).

| DISCUSSION
The purpose of this study was to examine the modulation of MEPs by SAI during fatiguing contractions in people with MS.A contraction protocol was employed that included a sustained low-intensity contraction, which is known to depress force generating capacity in both people with MS and healthy individuals.Following this sustained contraction, a low-intensity intermittent contraction protocol was used so that measurements of MEP and SAI could be made during the rest phases of each duty cycle.The main findings of this study were: (1) force, EMG and MEP responses were similar between groups when performing the contraction protocol, (2) SAI was significantly reduced throughout the contraction protocol and during recovery for the MS group compared to the healthy group and (3) no measure of inhibition was correlated to clinical scales of fatigue for the MS group.This study provides novel evidence that afferent inhibition differs in people with MS, where intracortical circuits appear to disinhibit SAI during fatiguing contractions.

| Depression in force generating capacity during the contraction protocol
The sustained 3-min 15% MVC caused comparable reductions in maximal force generating capacity in the MS group and the healthy control group.A decline in maximal force generating capacity, as observed here, is an accepted indicator that exercise-induced neuromuscular fatigue has occurred (Enoka & Duchateau, 2016;Gandevia, 2001;Kent-Braun et al., 2012).Within this context, the sustained low-intensity contraction reduced maximal force generating capacity of the APB by $28% for both groups, which suggests that the initial contraction task in the testing protocol induce similar effects on motor performance for each group.Subsequent to the sustained low-intensity contraction, a series of duty cycles were executed.Although sustained and intermittent contractions are known to elicit distinct physiological responses (Hunter, 2018), our primary objective was to conduct MEP and SAI measurements during each duty cycle while maintaining similar levels of force depression.Similar changes in maximal force and EMG between the groups allow us to determine if SAI contributes to the modulation of MEPs during fatiguing contractions in people with MS without needing to account for diseaserelated differences in motor output.

| SAI is reduced with MS
SAI reflects the inhibitory interactions between the S1 and M1 divisions of cortex.The strength of SAI is influenced by the amplitude of the sensory afferent volley, with greater inhibition associated with greater activity in sensory pathways.In the current study, there were no group differences detected for SSEP measurements.An absence of SSEP deficits opposes several previous MS studies (Chiappa, 1980;Comi et al., 1999;Dubbioso et al., 2022;Trojaborg & Petersen, 1979; Walsh  et al., 2005), where SSEP abnormality rates occurred in $60%-80% (Chiappa, 1980;Trojaborg & Petersen, 1979) of MS individuals.As there were no differences in the ascending SSEP in the current study, MS-related SAI differences were most likely associated with central integration of the sensory and motor signal.After completing the sustained 3-min 15% MVC, the MS group had 40% less inhibition of the MEP compared to the control group.
Similarly, there was consistently 15% less inhibition for the MS group throughout the subsequent intermittent contraction protocol.Given that MEPs were similar between groups when measured throughout the entire contraction protocol, we proposed that a disease mediated mechanism is present in people with MS, which attempts to maintain optimal levels of descending drive from the motor cortex.By disinhibiting inhibitory circuits, it may be possible to keep higher levels of facilitation of the MEP during fatiguing tasks (Hunter & Enoka, 2003;Klass et al., 2008;Søgaard et al., 2006;Taylor et al., 2016).Thus, similarities in MEPs may not necessarily indicate a preservation of M1 or corticospinal function in MS, but instead may reflect a compensation mechanism to enhance activity in motor circuits that regulate corticospinal activity.
Although this is the first study to reveal that fatiguing contractions suppress SAI, there are several previous reports that SAI is affected in people with MS while in a resting state (Cucurachi et al., 2008;Dubbioso et al., 2022;Mango et al., 2019;Topkan et al., 2022) or when administered pharmacological interventions known to improve motor function (Dubbioso et al., 2022;Nicoletti et al., 2019).The neurotransmitters that are implicated in SAI include acetylcholine and GABA, where SAI is enhanced by cholinergic transmission (Di Lazzaro et al., 2000, 2005;Fujiki et al., 2006) and reduced by GABAergic transmission (Di Lazzaro et al., 2007;Teo et al., 2009).Therefore, it is not surprising that disease modifying therapies such as dimethyl fumarate, an anti-inflammatory MS drug, which also stimulates acetylcholine and synaptic plasticity, increases SAI in people with MS (Nicoletti et al., 2019).It is important to note that an increase in SAI due to drug treatment does not necessarily align with our interpretations in the current study, as we propose that reductions in SAI are beneficial to MEP generation in people with MS.Thus, further research is required to determine if treatments, which improve specific symptom of MS (e.g.neuro-inflammation), have secondary consequences for muscle activation.With respect to GABAergic activity, our MS group exhibited lengthening of TMS-induced silent period in APB EMG throughout our fatiguing contraction task.The silent period is regulated by GABA B receptor activity and has an inhibitory effect on muscle activity (Hallett, 2000;Ziemann, 2004).The greater inhibition demonstrated with the MS silent period, and reduced inhibition demonstrated for SAI, highlights the complexity of inhibitory cortical circuits that are involved with muscle activation in people with MS.

| Clinical assessment of MS fatigue
Approximately 80% of individuals with MS report that fatigue is their most debilitating symptom.Unlike laboratory-based measurements, clinical assessment of fatigue will generally rely on the patient to provide selfassessments of their condition.The FSS is a self-report questionnaire, which provides a quantitative measure of the impact of fatigue on daily functioning, and the MFIS is a multidimensional tool that not only evaluates the severity of fatigue but also assesses its specific impacts on physical, cognitive and psychosocial aspects of daily life.Neither scales correlated to silent period or SAI in the MS group.Although it is tempting to suggest that these TMS measures do not relate to fatigue, selective correlations were identified for the silent period and SAI with clinical fatigue scales for the control group.Thus, it is possible that (1) the clinical scales are not sensitive enough to detect disease-related differences identified from lab-based measurements, and (2) the heterogeneity of disease impacts the perception of where fatigue originates for each individual.For example, individuals with more advanced physical disability may experience greater impacts on the physical subscale of the MFIS, while those with more cognitive involvement may report higher scores on the cognitive subscale.Nonetheless, the current study contributes to existing knowledge where an absence or correlation is often found between clinical fatigue scales and neurophysiological measures of neuromuscular fatigue in people with MS (Leodori et al., 2023;Mordillo-Mateos et al., 2019;Morgante et al., 2011;Perretti et al., 2004).These findings illustrate the importance of implementing multiple techniques in addition to clinical fatigue scales when quantifying fatigue in individuals with MS.

| Considerations
The current experiment employed hand-held TMS procedures, so the focal point accuracy of stimulating the motor representation of the APB is limited to the steadiness of the investigator.In addition, if head movement caused the coil to move to a different cortical area, it is unlikely that the unconditioned MEP would remain as stable as it did for both groups throughout the contraction protocol (Figure 5a; no main effect of time).The MS group also had $50% reduction in SAI compared to the control group, which we contend is too large to be a factor of coil misalignment.Once again, SAI measurements were stable through the contraction protocol for both the MS group and the control group.It is important to note that a larger number of stimulations are typically used to provide measures of SAI; however, any SAI measurements obtained outside of very brief post-contraction windows are very unlikely to reflect the effect that central fatigue has on intracortical processes.Indeed, SAI returned to baseline levels for both groups 6 min after completing the current contraction protocol, and our previous work has demonstrated that MEP area can return to baseline levels within 15 s following strong fatiguing contractions of the abductor digiti minimi (Dempsey & Kavanagh, 2021).
Although our stimulation techniques are ambitious, we believe that it is important to attempt experiments that invoke fatigue in people with MS, as fatigue is the most reported disability in this cohort.Our interpretations also assume that SAI reflects cortical involvement in MS, and not spinal inhibition.While some SAI studies have used H reflexes to verify the involvement (or lack of involvement) in spinal networks, it is also noted in these studies that it is not possible to reliably obtain H reflexes in hand muscles (Tokimura et al., 2000).An alternative approach in healthy individuals is to assess H reflexes in a remote muscle (Tokimura et al., 2000).However, this may not inform measures of SAI in MS as cortex and spinal neurons may have different degrees of dysfunction depending on the locus and severity of lesions.Future TMS research should also incorporate the MS Expanded Disability Status Scale to determine if clinical assessments of disability, and the location of motor dysfunction, align with dysfunction in motor circuits in the CNS.

| Conclusions
Approximate 80% of people with MS report that fatigue is the most disabling symptom of their condition.Therefore, understanding mechanisms of exercise-induced fatigue in MS may assist in understanding why symptoms fatigue-related symptoms emerge in this population.In the current study, MEP amplitude remained consistent between people with MS and healthy controls during the performance of a fatiguing contraction protocol.The modulation of MEP amplitude with SAI was reduced in the MS group, which suggests that intracortical circuits may disinhibit SAI in people with MS during fatiguing contractions.

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I G U R E 3 EMG and responses to motor cortical stimulation during 15% maximal voluntary contraction (MVC) measures obtained during the contraction and recovery phases.Abductor pollicis brevis (APB) root mean square (RMS) EMG (a) motor evoked potential (MEP), (b) peak-to-peak amplitude and MEP silent period (c) obtained during a 3 min sustained contraction, throughout duty cycles and in recovery.APB RMS EMG, MEP and silent period are expressed relative to measures taken during 15% MVC baseline contractions.Hash symbol indicates a main effect of time, and cross symbols indicate a group Â time interaction (p < .05).All data are presented as mean ± SD (MS group n = 14, control group n = 10).
T A B L E 2 Correlations between fatigue scales and TMS-related inhibition measures.Data are presented as the bivariate correlation coefficient (r values).Values in brackets indicate p-values, with asterisks indicating a significant correlation (p < .05).Abbreviations: FSS, fatigue severity scale; MFIS, modified fatigue impact scale; SAI, short-latency afferent inhibition; TMS, transcranial magnetic stimulation. Note: