Received: 10 February 2023 Revised: 20 March 2023 Accepted: 22 March 2023

DOI: 10.1002/brb3.2996

ORIGINAL ARTICLE

Investigating interindividual variability in corticomotor

reorganization during sustained hamstring pain: A randomized
experimental study

Rocco Cavaleri1 Jawwad Imam1 Ebonie Rio3 Nadia Moukhaiber1

Daniel Thomson1 Ariane Suhood1 Simon J. Summers1,2

1
Brain Stimulation and Rehabilitation
(BrainStAR) Lab, School of Health Sciences,
Western Sydney University, Sydney, New
South Wales, Australia
2
School of Biomedical Sciences, Queensland
University of Technology, Brisbane,
Queensland, Australia
3
School of Allied Health, La Trobe University,
Melbourne, Victoria, Australia
Correspondence
Rocco Cavaleri, School of Health Sciences,
Western Sydney University, Locked Bag 1797,
Penrith 2751, NSW, Australia.
Email: R.Cavaleri@westernsydney.edu.au
Abstract
Background: Increasing evidence suggests that pain drives maladaptive corticomotor
changes that may increase susceptibility to injury and promote symptom recurrence.
However, few studies have evaluated the influence of interindividual corticomotor
responses to musculoskeletal pain. Existing research in this area has also been lim-
ited largely to the upper limb. This is a pertinent point, given the functional and
neurophysiological differences between upper and lower limb muscles, as well as
the fact that most acute sporting injuries occur in the lower limb. Accordingly, this
study explored the variability of corticomotor responses to experimentally-induced
sustained hamstring pain and whether specific patterns of corticomotor reorganiza-
tion were associated with poorer outcomes (mechanical sensitivity, pain, or functional
limitation).
Method: Thirty-six healthy individuals participated. Following random allocation on
Day 0, the experimental group performed an eccentric exercise protocol of the
right hamstring muscles to induce delayed onset muscle soreness. The control group
performed repetition-matched concentric exercise that did not induce soreness. Mea-
sures of mechanical sensitivity, pain, function, and corticomotor organization were
collected at baseline and on Day 2.
Results and conclusions: Corticomotor responses to sustained hamstring pain were
variable. Individuals who developed corticomotor facilitation in response to hamstring
pain experienced greater mechanical sensitivity than those who developed corticomo-
tor depression. These novel data could have implications for rehabilitation following
lower limb pain or injury.

KEYWORDS
Hamstring, TMS, Pain, Corticomotor

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2023 The Authors. Brain and Behavior published by Wiley Periodicals LLC.

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https://doi.org/10.1002/brb3.2996

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1 INTRODUCTION
Accumulating evidence suggests that pain from musculoskeletal
injuries drives maladaptive corticomotor changes that may promote
symptom recurrence (Schabrun et al., 2015; Tsao et al., 2008, 2011).
Through the use of transcranial magnetic stimulation (TMS), several
studies have identified changes in corticomotor activity in response
to pain, characterized by shifts in the cortical representations of
affected muscles (Schabrun et al., 2017; Te et al., 2017) and changes
in corticomotor excitability (Mhalla et al., 2010; Salerno et al., 2000;
Strutton et al., 2005; Te et al., 2017). These corticomotor changes
have been associated with pain intensity and functional limitation
(Cavaleri et al., 2021; Seminowicz et al., 2019; Tsao et al., 2008).
Maladaptive changes in corticomotor pathways are hypothesized to
contribute toward injury recurrence by promoting abnormal move-
ment patterns that detrimentally alter soft tissue loading (Hodges &
Tucker, 2011). Observations of neuromuscular inhibition and deficits
in proprioceptive processing following musculoskeletal injury support
potential cortical adaptations to acute and chronic pain (Buhmann
et al., 2022; Fyfe et al., 2013; Summers et al., 2021).
Specific patterns of pain-induced corticomotor reorganization may
be associated with poorer outcomes over time. Indeed, a recent
review demonstrated that individuals displaying reduced corticomo-
tor excitability in response to acute experimental pain (lasting minutes)
report lower pain severity than those showing increased excitability
(Chowdhury et al., 2022). However, reduced excitability was associ-
ated with greater pain severity in individuals with sustained pain (lasting
days-to-weeks), suggesting that persistence of this adaptation may be
implicated in symptom chronicity or recurrence (Chowdhury et al.,
2022). Such data suggest that maladaptive corticomotor reorganiza-
tion may be a novel risk factor that could be targeted to attenuate pain
and reduce risk of reinjury.
While valuable, the exploration of corticomotor responses to mus-
culoskeletal pain has been limited primarily to the upper limb (Chowd-
hury et al., 2022). This is a pertinent point, given that up to 80% of acute
sporting injuries occur in the lower limb (Murphy et al., 2003). Evalua-
tion of lower limb corticomotor responses to acute pain is also essential
given the functional and neurophysiological differences between upper
and lower limb musculature (Kesar et al., 2018). Hand representa-
tions, responsible for fine motor control, are larger, more excitable, and
have greater connectivity with structures promoting endogenous opi-
oid release than proximal and lower limb representations, which drive
gross motor functions (Andre-Obadia et al., 2018; Palmer & Ashby,
1992). The capacity for corticomotor reorganization may therefore
differ between these regions. Indeed, region-specific differences in
corticomotor responses are supported by work demonstrating that
reorganization following upper limb training is graded from proximal to
distal, with hand representations showing greater reorganization than
shoulder representations (Krutky & Perreault, 2007).
Here, we explored the variability of corticomotor responses to
experimentally-induced sustained pain and whether specific patterns
of reorganization were associated with poorer outcomes (increased
mechanical sensitivity, pain, or functional limitation). Pain was induced
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CAVALERI ET AL .
in the hamstring muscle as this region is one of the most commonly
injured in the lower limb, and hamstring strains are associated with
a high recurrence rate (Brooks et al., 2006; Hawkins et al., 2001;
Malliaropoulos et al., 2018; Orchard & Seward, 2002). We hypothe-
sized that sustained hamstring pain would elicit variable patterns of
corticomotor reorganization, with increases in corticomotor excitabil-
ity being associated with poorer outcomes.
2 METHOD
2.1 Participants
Thirty-six healthy participants (20 female, 16 male) were recruited
for this exploratory randomized controlled study. Participants were
recruited through advertisements on Western Sydney University
notice boards, social media pages, and the university Physiotherapy
program website. A snowball approach was then utilized to broaden
the sample and identify additional volunteers.
Individuals were eligible for inclusion if they were aged greater than
18 years and were right-foot dominant as determined by the Waterloo
Footedness Questionnaire (van Melick et al., 2017). Participants were
excluded if they presented with a history of a hamstring injury, other
major lower limb injuries or surgeries, contraindications to exercise
as determined through the Physical Activity Readiness Questionnaire
(Helmerhorst et al., 2012), or contraindications to TMS as determined
through the TMS Adult Safety Screen questionnaire (Rossi et al., 2011,
2020).
All participants received written and verbal descriptions of the
experiment and provided written informed consent prior to testing. All
procedures were approved by the local institutional Human Research
Ethics Committee (H14252) and performed in accordance with the
Declaration of Helsinki.
2.2 Experimental protocol
Participants attended two experimental sessions (Day 0 and 2). Partici-
pants were requested to refrain from exercise 48 h prior to testing and
throughout the duration of the study. On Day 0, demographic (age, sex,
height, and weight) and affective-emotional (Depression, Anxiety, and
Stress Scale [DASS-21]; Osman et al., 2012) data were collected. Base-
line mechanical sensitivity (pressure pain thresholds [PPTs]), pain (area,
intensity, muscle soreness), functional (lower extremity functional
scale [LEFS] (Binkley et al., 1999), single-leg hop test, maximal vol-
untary isometric contraction [MVIC]), and corticomotor organization
(TMS maps) outcomes were also collected.
Randomization to either an eccentric (pain-provoking) (n = 26) or
concentric (non-pain-provoking) (n = 10) exercise protocol was per-
formed by an independent assessor using a random number generator
before testing on Day 0. A larger sample was recruited for the eccentric
group to enable sufficient exploration of interindividual variability in
response to sustained hamstring pain. Allocation was concealed using

CAVALERI ET AL .
consecutively numbered opaque envelopes. Participants completed
their allocated exercise program at the end of the session on Day 0. The
participants were requested to refrain from treating their pain until the
outcome measures were reassessed on Day 2. This included refraining
from administering any form of analgesics, lower limb exercise, or using
adjunct treatment modalities for pain such as heat, ice, or massage.
2.3 Experimentally-induced pain protocol
2.3.1 Experimental group: eccentric (pain-inducing)
exercise protocol
Twenty-six individuals were included in the experimental (pain-
inducing eccentric exercise) group. The eccentric protocol was con-
ducted using a Biodex isokinetic dynamometer (Biodex Multi-Joint
System 3, Shirley, NY, USA). Participants were seated upright and
secured to the seat using straps over the shoulders and their lap,
with their hips and knees in 90◦ of flexion. The rotational axis of
the dynamometer was aligned with the lateral femoral condyle of the
right leg. Immediately following a warm-up (3 sets of 10 eccentric
hamstring contractions, with each set gradually decreasing in veloc-
ity [90, 75, and 60◦ /s, respectively]), participants completed 10 sets
of 10 maximal eccentric contractions of the right hamstring muscles
through a 90◦ knee flexion range of motion and an angular veloc-
ity of 45◦ /s. The participants were instructed to resist maximally as
the lever arm pushed their leg into knee extension. As the lever arm
returned back to the start position (0◦ –90◦ ), the participants were
asked to relax and allow their leg to passively return to the starting
position (Johansson et al., 2007). Participants rested for 30 s after
each set. Standardized verbal encouragement was provided by the
investigators. Previous research has shown that such protocols induce
delayed onset muscle soreness (DOMS) that develops within 24 h and
peaks between 48 and 72 h postexercise (Armstrong, 1984). Follow-
ing each set, participants quantified their exertion levels using a rating
of perceived exertion (RPE) scale from 0 (no exertion) to 10 (maximal
exertion).
2.3.2 Control group: concentric (pain-free)
exercise protocol
Ten individuals were included in the control (pain-free concentric exer-
cise) group. Participants were seated upright on the dynamometer and
secured with their hips in 90◦ of flexion and knees in 0◦ of flexion.
Immediately following a warm-up (3 sets of 10 concentric hamstring
contractions, with each set gradually decreasing in velocity [90, 75,
and 60◦ /s, respectively]), participants completed 10 sets of 10 max-
imal concentric contractions of the right hamstring muscles through
a 90◦ knee flexion range of motion and an angular velocity of 45◦ /s.
The participants were instructed to maximally bring their knee into
flexion against the lever arm. As the lever arm returned back to the
start position, the participants were asked to relax and passively allow
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3 of 12
the knee to straighten (Johansson et al., 2007). A concentric protocol
accounts for the effort and fatigue experienced by the experimental
(pain) group, without inducing DOMS or changes in mechanical sen-
sitivity (Armstrong, 1984; Pérez et al., 2023). Participants rested for
30 s after each set. Following each set, participants quantified their
exertion levels using a RPE scale from 0 (no exertion) to 10 (maximal
exertion).
2.4 Assessments
All mechanical sensitivity, pain, and functional outcomes were col-
lected by the same investigator, as were all TMS-derived corticomotor
maps. All assessors were blinded to group allocation.
2.4.1 Mechanical sensitivity
Pressure pain threshold
PPT was assessed over the biceps femoris muscle belly, located halfway
between the ischial tuberosity and the lateral epicondyle of the tibia
(Hermens et al., 2000). The location of the muscle belly was confirmed
via palpation by an experienced physiotherapist. Force was applied per-
pendicularly to the skin using a handheld algometer (Somedic, 1 cm2
probe) and gradually increased at a rate of 30 kPA/s. The PPT was
defined as the point at which the sensation of pressure was first
reported to become a sensation of pain (De Martino et al., 2018; Rocha
et al., 2012). Three measures were taken and the mean PPT recorded.
This measure has been shown to have excellent test–retest reliability
(ICC 0.94) (Balaguier et al., 2016; Jones et al., 2007).
2.5 Pain and muscle soreness
2.5.1 Pain intensity
Participants scored their hamstring pain on an 11-point numerical rat-
ing scale (where 0 = no pain, and 10 = the worst pain imaginable) at
rest, as well as reaching for the floor with knees straight and during the
hamstring-drag test (Zeren & Oztekin, 2006). The hamstring-drag (or
“taking off the shoe”) test is a provocative assessment with a high sensi-
tivity and specificity for biceps femoris strain (Zeren & Oztekin, 2006).
2.5.2 Muscle soreness
Muscle soreness was assessed using a modified 7-point Likert scale
with the following categories: 0 = “A complete absence of soreness,”
1 = “A light soreness in the muscle felt only when touched/a vague
ache,” 2 = “A moderate soreness felt only when touched/a slight
persistent pain,” 3 = “A light muscle soreness when walking up and
down stairs,” 4 = “A light muscle soreness when walking on flat sur-
face,” 5 = “A moderate muscle soreness, stiffness, or weakness when

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walking,” 6 = “A severe muscle soreness, stiffness, or weakness that
limits my ability to move.” This scale has been used in previous studies
investigating muscle soreness in the hamstring muscles (Gibson et al.,
2006).
2.5.3 Pain area
Pain area was assessed using a body chart on which participants were
asked to mark all areas of pain. These charts were later digitized and
imported into raster graphics software (Photoshop 6.0; Adobe, San
Jose, CA, USA) where the selected areas were isolated and a pixel count
determined (Cavaleri et al., 2019).
2.6 Functional measures
2.6.1 Lower extremity functional scale
The LEFS was used to assess the functional status of the lower limb.
The scale presents a series of everyday activities requiring the lower
limb, with the participant recording a score from 4 (“no difficulty”) to
0 (“extreme difficulty or unable to perform”) for each item. The final
score is summed, and a higher score (maximum of 80) represents lower
levels of disability and greater function. The LEFS scale has excellent
test–retest reliability (ICC 0.85–0.99) (Mehta et al., 2016).
2.6.2 Maximum voluntary isometric contraction
The MVIC was measured using a Biodex isokinetic dynamometer
(Biodex Multi-Joint System 3, Shirley, NY, USA). Participants were
seated upright, with their knees and hips in 90◦ of flexion. The par-
ticipants were required to do a set of three maximal isometric knee
flexions for 5 s each with 30 s rest in between each contraction (Dover
et al., 2012). The highest measurement (in N m) was used for analysis.
2.6.3 Single-leg hop test
The horizontal single-leg hop test provides insight into hamstring mus-
cle function and injury risk (van der Harst et al., 2007). All participants
were asked to perform the test barefoot, hopping forward as far as
possible with the right leg. Hallux–hallux distance (cm) was recorded
using a metric tape, and the greatest distance of three attempts was
recorded.
2.7 Corticomotor organization
Corticomotor organization was assessed using TMS mapping. This
technique is a noninvasive means by which to investigate the size, loca-
tion, and excitability of corticomotor representations (Cavaleri et al.,
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CAVALERI ET AL .
2015). During TMS, electromyography (EMG) is used to record the
muscle (biceps femoris) responses evoked following motor cortex stim-
ulation. All mapping procedures were reported in accordance with the
TMS-specific methodological assessment checklist (Chipchase et al.,
2012).
2.7.1 Electromyography
Surface EMG (Ag–AgCl, Noraxon dual electrodes, interelectrode dis-
tance 2.0 cm) was used to record motor evoked potentials (MEPs) fol-
lowing TMS over the biceps femoris corticomotor representation. The
active electrode was placed over the belly of the right biceps femoris
muscle, and the ground electrode was positioned over the anterior
superior iliac spine. EMG signals were amplified (×1000), bandpass
filtered (20–1000 Hz), and sampled at 2 kHz using a Power 1401
Data Acquisition System and Signal3 software (Cambridge Electronic
Design, Cambridge, United Kingdom).
2.7.2 Hotspot and active motor threshold
determination
During TMS, single-pulse, biphasic stimuli were delivered using a
Magstim Super Rapid2 Plus1 (Magstim Co. Ltd, Dyfed, UK) and a double
70 mm air-cooled figure-of-eight coil. The coil was placed tangentially
to the skull with the handle pointing posteriorly, inducing a current in
the posterior–anterior direction (Richter et al., 2013). Visual feedback
of EMG activity was provided via a monitor positioned at eye level 2 m
in front of the participant.
The “hotspot” was defined as the coil position that evoked a max-
imal peak-to-peak MEP in the target muscle at a given stimulation
intensity (Cavaleri et al., 2017a, 2017b; Ferreri & Rossini, 2013). The
active motor threshold (aMT) was defined as the minimum TMS inten-
sity required to elicit at least 5 discernible MEPs in a train of 10 stimuli
delivered to the hotspot during a submaximal hamstring contraction
(Groppa et al., 2012). A Brainsight stereotactic frameless neuronaviga-
tion system (Rogue Research Inc.) was used to determine the hotspot
and aMT.
2.7.3 TMS mapping protocol
During mapping, all stimuli were delivered, whereas the participant
maintained a submaximal contraction of the hamstring muscles (10%
of maximal EMG recorded during voluntary isometric knee flexion).
The stimulation intensity was set at 110% of the aMT identified at
baseline. Ninety stimuli were delivered pseudorandomly to the scalp
over a 5 × 7 cm grid (six rows and eight columns) oriented to the cra-
nial vertex (Cavaleri et al., 2018; Van De Ruit et al., 2015). The grid
was superimposed on a generic brain image in the neuronavigation dis-
play. Using this system, each participant’s cranial landmarks (nasion,
inion, tragi, eye canthi, and cranial vertex) were registered at baseline

CAVALERI ET AL .
and saved for use across sessions to ensure consistent grid placement,
neuronavigation, and targeting.
2.7.4 TMS map processing
Maps were generated offline using MATLAB using an approach
adapted from previous studies (Cavaleri, 2022; Cavaleri et al., 2018;
Van De Ruit et al., 2015). Briefly, triangular linear interpolation was
used to create a full surface map within a transformed plane containing
stimulation coordinates and their corresponding MEP amplitudes. The
resultant map was divided into 2500 partitions (50 × 50). Partitions
with MEP amplitudes exceeding 25% of a participant’s peak response
were labeled as “active.” To calculate map area, the number of active
partitions was divided by 2500 and multiplied by the size of the stimu-
lated area. Map volume was determined by summing the approximated
MEP amplitudes (in millivolts) of all active partitions in the matrix. The
center of gravity (CoG), or amplitude weighted center of the map, for
each muscle was calculated using the formula: CoG = Σ(xz)/Σz; Σ(yz)/Σz
(where x = mediolateral coordinate; y = anteroposterior coordinate;
and z = corresponding MEP amplitude). Shifts in the location of the
CoG were calculated as the Euclidean distance (ED) from baseline using
the formula: ED = √(y1 − y2)2 + (x1 − x2)2 , (where y = anteroposterior
coordinate; x = mediolateral coordinate; and 1 and 2 refer to baseline
and post-training values, respectively) (Cavaleri et al., 2018; Van De
Ruit et al., 2015).
2.8 Statistical analyses
All statistical analyses were performed using the Statistical Package for
the Social Sciences software (version 23; IBM Corp, Armonk, NY, USA).
Statistical significance was set at p < .05.
2.8.1 Effect of experimental hamstring pain on
mechanical sensitivity, pain, function, and
corticomotor outcomes
The effect of sustained experimental hamstring pain on mechanical
sensitivity (PPTs), pain (numerical rating scale, muscle soreness, pain
area), functional (LEFS, MVICs, single-leg hop test), and corticomotor
(map volume, map area, CoG displacement) outcomes was analyzed
using mixed-model ANOVAs with factors “group” (experimental vs.
control) and “time” (Baseline and Day 2 postexercise). The Shapiro–
Wilk test and Mauchly’s test of sphericity were applied to assess
assumption of normality and sphericity, respectively (Moulton, 2010;
Shapiro & Wilk, 1965). The Greenhouse–Geisser correction for non-
sphericity was applied for datasets that violated the assumption of
sphericity (Abdi, 2010). Where appropriate, post hoc analyses were
performed by using Sidak-adjusted multiple comparison tests.
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5 of 12
2.8.2 Variability in corticomotor responses to
experimental hamstring pain
Previous studies have found that corticomotor reorganization in
response to pain has high interindividual variability, with people
displaying either a decrease in corticomotor excitability (“depres-
sion”) or an increase (“facilitation”) that could affect pain experi-
ences (Cavaleri et al., 2021; Seminowicz et al., 2019). Participants
in the experimental group were therefore categorized as “depres-
sors” or “facilitators” in terms of both their map volume and map
area changes during hamstring pain. To do so, the mean absolute
% changes in map volume (Δvolume) and map area (Δarea) from
baseline in the control group were determined. Participants in the
experimental group were classified as facilitators if, at Day 2 pos-
texercise, their mean % change in map volume or map area from
baseline exceeded the Δvolume or Δarea in the control group by
greater than 1 standard deviation (SD) (Cavaleri et al., 2020). Con-
versely, participants were classified as depressors if their change
in map volume or area was at least 1 SD below the Δvolume or
Δarea in the control group. All other participants were classified as
nonresponders.
2.8.3 Relationship among corticomotor
reorganization, pain, and function
To determine whether facilitators in the experimental group exhib-
ited different mechanical sensitivity, pain, and function changes to
depressors, repeated-measures ANOVAs were conducted with the
between-subject factor “Response” (facilitators vs. depressors) and the
within-subject factor “time” (Baseline and Day 2 postexercise). Nonre-
sponders were excluded from this analysis. Pearson’s correlations were
run to further explore the relationship between changes in corticomo-
tor outcomes and changes in mechanical sensitivity, pain, and function.
The following values were used to interpret Pearson’s correlation
coefficient: r < 0.3 = weak correlation; r between 0.3 and 0.5 = mod-
erate correlation; and r > 0.5 = strong correlation (Laerd Statistics,
2020).
3 RESULTS
3.1 Participant characteristics
Participant characteristics are summarized in Table 1. The experi-
mental (eccentric) and control (concentric) groups were comparable
on all baseline variables. All participants attended the sessions as
intended and no adverse events were reported. There was no signifi-
cant difference in RPE between groups over the course of the exercise
protocols (time: F3.5, 119.1 = 40.06, p < .01; group: F1, 34 = 0.01, p = .94;
group × time: F3.5, 119.1 = 1.05, p = .38).
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6 of 12 CAVALERI ET AL .

TA B L E 1 Participant characteristics.

Experimental group mean (SD) Control group mean (SD) p Value

Sample size (n) 26 10 –
Sex (male, female) 11, 15 5, 5 –
Age (years) 22 (4) 23 (4) .58
Height (cm) 168.5 (9.6) 169.9 (6.6) .67
Weight (kg) 72.3 (19.6) 70.8 (12.3) .83
Baseline aMT (%) 85 (13) 78 (17) .18
DASS-21 depression 1.9 (2.6) 2.3 (3.6) .73
DASS-21 anxiety 2.0 (2.1) 2.1 (2.1) .90
DASS-21 stress 3.0 (3.0) 2.1 (3.0) .41

Abbreviations: aMT, active motor threshold; cm, centimeters; DASS-21, depression anxiety stress scale; kg, kilograms; n, number; SD, standard deviation.

TA B L E 2 Differences in pain outcomes between groups.

Mean (SD) values on Day 2

Outcomes Eccentric Concentric t df MD (95% CI) p Value
Pain (NRS)
-At rest 1.3 (1.2) .0 (.0) 3.37 34 1.3 (0.5–2.1) <.01*
Reaching for floor 5.4 (2.5) .2 (.4) 6.55 34 5.2 (3.6–6.8) <.01*
Hamstring-drag test 3.4 (2.1) .0 (.0) 4.87 34 3.4 (2.0–4.8) <.01*
Muscle soreness 4.5 (1.8) .2 (.4) 7.20 34 4.3 (3.1–5.5) <.01*
Pain area 25.9 (16.6) .2 (.4) 4.85 34 25.7 (14.9–36.4) <.01*

Abbreviations: CI, confidence interval; df, degrees of freedom; MD, mean difference; NRS, numerical rating scale; SD, standard deviation.
*p < .05.

3.1.1 Effect of experimental hamstring pain on
mechanical sensitivity, pain, and function
There was no significant difference between the experimental (pain-
inducing eccentric exercise) and control (pain-free concentric exercise)
groups in terms of mechanical sensitivity, assessed using PPTs (time:
F1, 34 = 0.01, p = .78; group: F1, 34 = 1.76, p = .91; group × time:
F1, 34 = 2.41, p = .13). However, the experimental group had greater
pain at rest (p < .01), when reaching for the floor with knees
straight (p < .01), and during the hamstring-drag test (p < .01)
compared to the control group on Day 2 (Table 2). As shown in
Table 2, the experimental group also reported greater muscle sore-
ness (p < .01) and a larger pain area (p < .01) than the control
group.
There were also differences between groups in terms of function
following the exercise protocol. A significant group × time interac-
tion was observed for LEFS scores (time: F1, 34 = 29.72, p < .01;
group: F1, 34 = 20.93, p < .01; group × time: F1, 34 = 20.93, p < .01).
Post hoc analyses revealed that LEFS scores in the experimental
group (51.4 ± 17.5) were lower than those of the control group
(77.5 ± 5.9) on Day 2, reflecting poorer lower limb function (p < .01).
There were also differences between groups in MVIC recordings over
time (time: F1, 34 = 11.58, p < .01; group: F1, 34 = 2.67, p = .11;
group × time: F1, 34 = 25.87, p < .01). Specifically, the experimental
group had lower MVIC recordings (60.8 ± 41.2) than the control group
(103.2 ± 41.2) (p < .01) on Day 2. However, there was no difference
between groups in single-leg hop performance (time: F1, 34 = 0.18,
p = .68; group: F1, 34 = 1.31, p = .26; group × time: F1, 34 = 1.13,
p = .72).
3.1.2 Effect of experimental hamstring pain on
corticomotor organization
There were no overall changes in map area (time: F1, 34 = 0.18, p = .68;
group: F1, 34 = 3.35, p = .08; group × time: F1, 34 = 0.14, p = .71)
or volume (time: F1, 34 = 0.10, p = .76; group: F1, 34 = 3.68, p = .06;
group × time: F1, 34 = 0.13, p = .72) in either the experimental or control
groups, suggesting no predictable change in corticomotor excitabil-
ity in response to acute experimental hamstring pain. However, there
were significant differences in CoG displacement, reflective of cortico-
motor reorganization, between groups (t34 = 2.73, p = .01). Specifically,
the experimental group demonstrated greater CoG displacement from
baseline (1.2 ± 0.8) than the control group (0.4 ± 0.1). Figure 1 presents
a summary of the changes in TMS mapping outcomes over time for each
group.

CAVALERI ET AL .
F I G U R E 1 Changes in transcranial magnetic stimulation (TMS) mapping outcomes (A: Map Area, B: Centre of Gravity, C: Map Volume) over
time. Gray line: control group, black line: experimental group, cm: centimeters, mV: millivolts.
3.2 Variability in corticomotor responses to
experimental hamstring pain
Changes to the representation of biceps femoris in response to sus-
tained experimental pain were variable. Indeed, the mean absolute
Δvolume ± SD in the experimental group was 177% ± 364%, and the
mean absolute Δarea ± SD was 87% ± 130%. Conversely, the control
group exhibited little variability in corticomotor responses over time.
The mean absolute Δvolume ± SD in the control group was 9% ± 3%,
and the Δarea + SD in this group was 12% ± 7%.
Based upon the criteria outlined in Section 2, 12 (46%) participants
in the experimental group were classified as facilitators, 12 (46%) as
depressors, and 2 (8%) as nonresponders in terms of map volume. In
terms of map area, 10 (39%) participants in the experimental group
were classified as facilitators, 11 (42%) as depressors, and 5 (19%)
as nonresponders. Center of gravity displacement ranged from 0 to
3 cm. All participants in the control group were considered to be
“nonresponders.”
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7 of 12
3.3 Relationship among corticomotor
reorganization, pain, and function
As shown in Table 3, a strong inverse correlation between map volume
and PPTs was identified.
There was a significant map volume response × time interaction
for PPTs (time: F1, 22 = 2.73, p = .11; group: F1, 22 = 0.33, p = .57;
group × time: F1, 24 = 7.33, p = 0 < .01). Post hoc analyses revealed
that individuals who demonstrated increases in map volume (cortico-
motor facilitation) had significantly lower PPTs (increased mechanical
sensitivity) on Day 2 than those demonstrating reductions in map vol-
ume (corticomotor depression) (p = .04) (Figure 2). Similarly, increases
in map area during experimental hamstring pain were moderately-
to-strongly correlated with reductions in PPTs. A repeated measures
ANOVA revealed a significant map area response × time interaction
for PPTs (time: F1, 19 = 2.87, p = .11; group: F1, 19 = 0.48, p = .50;
group × time: F1, 19 = 13.95, p < .01), with individuals demonstrat-
ing increases in map area possessing lower PPTs on Day 2 than those
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8 of 12 CAVALERI ET AL .

TA B L E 3 Correlations among corticomotor reorganization, pain, and function.

Correlation coefficient (p value)

Pain at rest Reaching for Hamstring Muscle
PPT (NRS) floor (NRS) drag (NRS) Pain area soreness LEFS MVC SLH
Map area −.46 (.02)* .05 (.79) .06 (.77) .16 (.42) .25 (.22) .14 (.49) −.16 (.45) −.02 (.94) −.31 (.12)
Map vol. −.56 (<.01)* .07 (.72) .21 (.30) .14 (.48) .22 (.28) .17 (.40) −.20 (.32) −.05 (.80) −.17 (.40)
CoG disp. .22 (.29) −.16 (.44) −.36 (.07) −.04 (.86) −.08 (.71) −.32 (.11) −.04 (.84) −.28 (.17) −.03 (.88)

Abbreviations: CoG disp., center of gravity displacement; LEFS, lower extremity functional scale; MVIC, maximal voluntary isometric contraction; NRS,
numeric rating scale; PPT, pressure pain threshold; SLH, single-leg hop.
*p < .05.

The variable corticomotor changes observed in response to sus-

F I G U R E 2 Difference in pressure pain thresholds (PPTs) between
facilitators and depressors. Gray line: depressors (map volume), black
line: facilitators (map volume), *p < .05.
demonstrating reductions in map area (p = .04). As shown in Table 3,
no further correlations between corticomotor outcomes and pain or
function were identified.
4 DISCUSSION
This was the first study to explore the variability of corticomotor reor-
ganization in response to sustained lower limb pain. Corticomotor
responses were variable, with 46% of participants displaying cortico-
motor facilitation, 46% displaying corticomotor depression, and 8%
being considered nonresponders based upon map volume. Further
investigation of this variability revealed that corticomotor facilitation
was strongly correlated with greater mechanical sensitivity (lower
PPTs). Corticomotor depression was correlated with lower mechanical
sensitivity (higher PPTs), suggesting a potentially protective response.
Corticomotor reorganization was not associated with changes in pain
intensity (numerical rating scale) or function. These data provide novel
insights into the potential mechanisms underlying recurrent hamstring
pain and injury.
tained hamstring pain are consistent with those reported in studies of
the upper limb. A recent systematic review and meta-analysis of 90
data points across five studies demonstrated that, relative to baseline,
57% of data points taken during sustained pain reflected decreased
corticomotor excitability and 43% reflected increased excitability. Such
variability in corticomotor responses is congruent with that observed
following both motor skill learning (Cavaleri et al., 2020; van de Ruit
& Grey, 2019) and noninvasive brain stimulation (Fratello et al., 2006;
Martin et al., 2006; Müller-Dahlhaus et al., 2008; Wiethoff et al., 2014).
Though yet to be completely elucidated, this variability is thought to be
driven by interindividual differences in factors such as anatomy (e.g.,
cortical thickness) (Ridding & Ziemann, 2010), kinesiophobia (Summers
et al., 2020), and history of exercise (Ridding & Ziemann, 2010). Prior
synaptic activity in a cortical region is also thought to influence sub-
sequent reorganization. For example, Siebner et al. (2004) found that
corticomotor responses to repetitive TMS can be manipulated by prior
administration of either anodal or cathodal transcranial direct current
stimulation. It is therefore plausible that an individual’s history of pain
and associated synaptic adaptations could influence their corticomotor
responses to subsequent pain experiences.
The relationships between corticomotor outcomes and mechanical
sensitivity observed in the present study contrast with that observed in
studies of sustained upper limb pain. Previous work has demonstrated
that corticomotor depression is associated with reduced upper limb
pain severity during pain lasting minutes-to-hours but increased pain
severity during sustained pain (lasting days-to-weeks) (Chowdhury
et al., 2022). Consistent with existing hypotheses (Hodges & Tucker,
2011), these studies suggest that corticomotor depression may be a
beneficial short-term adaptation to pain, but persistence of this adap-
tation may be associated with poorer long-term outcomes. However,
previous work examining corticomotor responses to sustained pain
has been limited to the upper limb, with a recent systematic review
acknowledging that region-specific corticomotor responses have yet to
be adequately explored (Chowdhury et al., 2022).
The present study extends existing literature by demonstrating that,
in contrast to findings from upper limb studies, corticomotor depres-
sion may represent a beneficial response during sustained hamstring
pain. However, it is important to note that, although corticomo-
tor depression was associated with improved mechanical sensitivity,
no such relationship was observed in terms of pain intensity (on a

CAVALERI ET AL .
numerical rating scale). The reason for this discrepancy is unclear, but it
should also be noted that PPTs reflect a distinct mechanism in periph-
eral neural sensitivity, whereas pain intensity is a subjective report that
may encapsulate a greater degree of psychosocial influences. Indeed,
previous work has demonstrated that pain intensity is not necessarily
correlated with areas of increased mechanical sensitivity in the lower
limb (Sánchez Romero et al., 2020).
It is reasonable to postulate that corticomotor adaptations to pain
could be region-specific. The upper limb is responsible for fine motor
tasks, so a reduction in corticomotor excitability during acute pain
in this region is thought to be beneficial as a means of restricting
movement and ensuring protection from further harm (Cavaleri et al.,
2021; Hodges & Tucker, 2011). In contrast, the lower limb is crucial to
locomotion, so an increase in corticomotor excitability could plausibly
facilitate escape from harm (Hodges & Tucker, 2011). Consistent with
contemporary theories of motor adaptation to pain (Hodges & Tucker,
2011), persistence of these early corticomotor responses could elicit
detrimental long-term consequences. Indeed, persistent increases in
quadriceps corticomotor excitability have been observed in individuals
with patellar tendinopathy (Rio et al., 2016) compared to healthy con-
trols. The relationship between increased sensitivity and corticomotor
facilitation observed in this study supports that, while potentially pro-
tective in the upper limb (Chowdhury et al., 2022), ongoing increases
in corticomotor excitability may contribute toward poorer outcomes
in lower limb pain or injury. However, further work exploring the tran-
sition from acute to sustained and chronic pain is required to confirm
these hypotheses.
4.1 Future directions and clinical implications
The results of this study are of potential clinical importance. Increasing
emphasis has been placed upon the need for prognostic indicators of
lower limb recovery or pain persistence (Sánchez Romero et al., 2021).
Our data raise the possibility that corticomotor facilitation in response
to hamstring pain could indicate susceptibility to greater mechanical
sensitivity and contribute to delayed return-to-play following ham-
string pain or injury. Indeed, pain on palpation (an index of mechanical
sensitivity) is used commonly to progress hamstring rehabilitation,
with higher pain sensitivity predictive of longer return-to-play times
after hamstring injury (Hickey et al., 2022). Higher mechanical sensi-
tivity may also impede engagement with the rehabilitation process,
potentially impacting hamstring recovery and reinjury risk. As such,
techniques aimed at restoring corticomotor excitability, and therefore
mechanical sensitivity, may aid the rehabilitation process following a
hamstring injury. Indeed, the use of noninvasive brain stimulation has
shown promise as a means of expediting recovery of experimental and
clinical musculoskeletal pain (Borovskis et al., 2021; Cavaleri et al.,
2019; Moisset et al., 2016; Moukhaiber et al., 2022; O’Connell et al.,
2018) and mechanical sensitivity has been suggested as a potential
marker for evaluating the success of such strategies (Pedersini et al.,
2020). However, given that numerical ratings of pain and function
in the present study were not impacted by changes in corticomotor
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9 of 12
excitability, these clinical implications remain speculative. Larger
datasets, in a hamstring-injured cohort, are required to confirm
the current findings and understand the impact of corticomotor
reorganization on injury prognosis and risk of reinjury.
This study has several strengths. A control group (concentric exer-
cise) was employed, mitigating the potential confounding effects of
repetition and fatigue on corticomotor outcomes. Assessors were also
blinded to minimize measurement bias. However, this study is not with-
out limitations. The results of this study are based on an experimental
pain model (DOMS) that mimics signs and symptoms associated with
hamstring injury (pain on palpation and loss of function). Whether our
results can be translated to clinical hamstring pain requires further
investigation. Another potential limitation of this study is that out-
come measures were not followed-up beyond the resolution of pain.
Previous studies have shown maintenance of corticomotor adapta-
tions following experimentally-induced pain in the upper limb (Burns
et al., 2016; Chowdhury et al., 2022). Exploring whether a similar phe-
nomenon occurs in response to hamstring pain would have important
implications for rehabilitation and long-term reinjury risk.
Finally, it is important to acknowledge that the reliability of
TMS mapping requires further exploration. Although the technique
employed has demonstrated excellent within- and between-session
reliability when assessing corticomotor representations of upper limb
(Cavaleri et al., 2018, 2019, 2021; Van De Ruit et al., 2015) and low back
(Cavaleri et al., 2020) musculature, the reliability of rapid TMS mapping
involving the lower limb is yet to be completely elucidated. Upper limb
muscles possess large, relatively superficial and excitable corticomo-
tor representations, meaning that they readily elicit consistent MEPs
(Palmer & Ashby, 1992; Wassermann et al., 1992). Conversely, lower
limb muscles have fewer corticospinal projections arising from a rela-
tively small and deep cortical area, making it more difficult to obtain
stable MEPs (Palmer & Ashby, 1992; Wassermann et al., 1992). Lower
limb muscles also have a greater proportion of ipsilateral projections
from M1 than upper limb muscles (Strutton et al., 2004). The reliability
of map recordings may therefore differ between these regions. Accord-
ingly, it is plausible that a degree of the variability observed throughout
this study was attributable to methodological factors. However, given
the minimal variability observed over time in the control group (mean
absolute Δvolume < 10% in the control group vs. Δvolume > 175% in
the experimental group), it is valid to suggest that most of the variabil-
ity observed in the experimental group was attributable to pain rather
than being a product of methodological factors or time.
5 CONCLUSION
This study is the first to explore the variability of corticomotor
responses to experimentally-induced sustained hamstring pain. In con-
trast to findings from the upper limb, individuals who developed
corticomotor facilitation in response to hamstring pain experienced
greater mechanical sensitivity than those who developed corticomotor
depression. These novel data could have implications for rehabili-
tation following hamstring pain or injury. Further work in clinical

10 of 12
populations is required to determine whether interindividual changes
in corticomotor outcomes impact prognosis and risk of reinjury.
AUTHOR CONTRIBUTIONS
Rocco Cavaleri, Simon J. Summers, and Ebonie Rio were all involved
in the design, writing, and editing of the study and manuscript. Rocco
Cavaleri, Syed Jawwad Imam, Nadia Moukhaiber, Daniel Thomson, and
Ariane Suhood were all involved in data collection. The final manuscript
was approved by all authors.
ACKNOWLEDGMENTS
Open access publishing facilitated by Western Sydney University, as
part of the Wiley - Western Sydney University agreement via the
Council of Australian University Librarians.
CONFLICT OF INTEREST STATEMENT
Dr Ebonie Rio, a senior research fellow, NHMRC, was funded as an
early career researcher. There are no conflict of interests or additional
acknowledgments to report.
DATA AVAILABILITY STATEMENT
Individual-level data that support the findings of this study are avail-
able from the corresponding author, Rocco Cavaleri, upon reasonable
request.
ORCID
Rocco Cavaleri https://orcid.org/0000-0001-9499-1703
PEER REVIEW
The peer review history for this article is available at https://publons.
com/publon/10.1002/brb3.2996.
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