The Influence of Nociception on Exercise Induced Fatigue: the Analgesic Role of Acute Exercise and Long-term Training of Different Exercise Modalities
Phase: NA
Conditions: Training, Exercise, Fatigue, Occlusion
Sponsor: Faculdade de Motricidade Humana

 
 
 
 
 
 
 
The influence of nociception on exercise induced fatigue: the analgesic 
role of acute exercise and long-term training of different exercise 
modalities 
 
 
Doutorando: André Dias Gonçalves, MSc, LabFNM, CIPER 
 
Orientador: Gonçalo Vilhena de Mendonça, PhD, LabFNM, CIPER 
 
Afiliação: Laboratório de Função Neuromuscular (LabFNM), Centro 
Interdiscplinar para o Estudo da Performance Humana (CIPER), Faculdade 
de Motricidade Humana - Universidade de Lisboa 
 
 
Last Update: 30th may 2021

1. Introduction 
 
1.1 Nociception and Health 
The International Association for the Study of Pain defines pain as an unpleasant 
sensory and emotional experience associated with actual or potential tissue damage, 
partially related to the magnitude of the nociceptive signal (1). 
Important reductions in nociception are often observed after 8 to 12 weeks of 
exercise (2); however, as little as 1 session of exercise can induce hypoalgesia (3, 4). This 
phenomenon is known as exercise-induced hypoalgesia (EIH), a generalized decrease in 
nociception sensitivity that occurs during exercise and for a short time afterwards (<30 min), 
relevant in minimizing symptoms in persons with chronic pain (5). Nociception has been 
quantified in a variety of ways in studies of EIH, with quantitative sensory testing most 
often used. Quantitative sensory testing describes a series of tests that measure the 
perceptual responses to systematically applied sensory stimuli (usually pressure, thermal, 
or electrical) (6). These tests typically involve the quantification of the individual 
nociceptive threshold and tolerance, which are, respectively, the minimum intensity of a 
stimulus that is perceived as painful and the maximum intensity to a noxious stimulus that 
the participant is willing to tolerate (7). These measures are frequently assessed in the 
immediate post-exercise period (e.g., 0–15 minutes after exercise termination). 
The magnitude of EIH appears to vary according to the combination of volume and 
intensity of exercise (8, 9, 10), the type of noxious stimulus used (3), the method used to 
quantify nociception (e.g. threshold, tolerance, rating) (3), the site (e.g. over an exercised or 
non-exercised muscles) (11, 12, 13), and the timing of nociception assessment (e.g. during 
or following exercise) (14, 15, 16). Analgesic effects are most common seen when noxious 
mechanical stimuli are used rather than thermal (17) and its effects are more pronounced in 
local than in remote muscles (12). While aerobic and isometric resistance exercise have 
been extensively investigated and its analgesic effect are relative unequivocal, dynamic 

resistance exercise has been far less studied (18). In addition, to our knowledge, only one 
study verified the hypoalgesic effect of a 30s Wingate anaerobic exercise (19).   
The most commonly proposed mechanism of EIH relies on enhanced descending 
inhibition by activation of the opioid and cannabinoid systems (5, 18, 20). The contraction 
of skeletal muscle increases the discharge of mechanosensitive afferents (i.e., A-delta and 
C fibers) which, in turn, activate central descending opioid analgesic pathways (21, 22). 
Exercise also increases the release of endogenous cannabinoids (23). The endocannabinoid 
system is a neuromodulatory system composed of cannabinoid receptors (CB1, CB2), their 
endogenous ligands, that is, the endocannabinoids (Narachidonylethanolamine [AEA] and 
2-arachidonoylglycerol [2-AG]), and proteins responsible for their metabolism (24). The 
presence of cannabinoid receptors in nociceptive-processing areas of the brain and spinal 
cord suggests that endocannabinoids contribute to the control of pain through the activation 
of CB1 receptors (24). The circulating levels of endocannabinoids increase after isometric 
exercise (23). These opioid and cannabinoids pathways have receptors not only throughout 
the central nervous systems but also at the periphery and can produce analgesia when 
stimulated (18). Furthermore, there is evidence of an interplay between the endocannabinoid 
and opioid systems, such that the activation of one system is mediated by the other system 
(25).  
Exercise-induced changes in the cardiovascular system have also been proposed as 
a mechanism of EIH (18). Exercise causes an increase in blood pressure which results in the 
activation of baroreceptors, innervated by vagal afferents, that are synaptically connected to 
pain modulatory brain areas, associated with blood pressure control, that decrease pain 
sensibility when activated (26, 27). This network is thought to be composed of 
interconnected areas including the insular and prefrontal cortices, the amigdala, the 
hypothalamus, the periaqueductal gray, the nucleous tractus solitarius. However, there’s still 
little evidence to support this (28, 29), once acute increases in blood pressure by exercise 
could not account for the persistence of EIH after exercise (i.e. 15 min after exercise 

cessation blood pressure would have presumably returned to baseline values, or indeed be 
lower) (18). 
The influence of exercise on reducing the sensitivity of the central nervous system 
has also been explored as a mechanism of EIH (18). There is evidence that acute exercise 
can reduce temporal summation (30, 31). Very few studies in humans have permitted 
description of the sites in the central nervous system from which EIH arises. Non-invasive 
neurophysiological techniques, using functional magnetic resonance imaging (FMRI) and 
electroencephalographic (EEG) event related potentials (ERP), can provide some insights 
(18). Ellingson et al., (2016) (32), with FMRI, found that cycling activated brain areas, like 
the left dorsolateral prefrontal lobe of the anterior insula, that are involved in descending 
pain pathways inhibition, decreasing pain sensitivity. More recently, Zheng et al. (2021) 
(33) and Wu et al. (2022) (34), using heat-evoked brain responses, provided evidence that 
cycling at 70% of heart rate reserve for 20 min and elbow flexor isometric contraction at 
40% MVC for 3min, respectively, induced the reduction of N2-P2 peak-to-peak amplitudes 
(time domain) as well as enhanced alpha oscillation power (frequency domain) in both 
exercised and non-exercised limbs (corroborated by pressure and heat pain thresholds 
increases in local and distal muscles). The N2-P2 potentials mainly originates from the 
anterior and medial cingulate gyrus and reflect the cognitive and emotional processing of 
pain perception; it´s amplitude reveals subject´s pain perception. Alpha oscillation power 
reflects the activation of the resting state sensory-motor neural network and reflects the 
central descending inhibitory function. Therefore, attending the global EIH effect, the 
authors concluded that it could be related not only to the modulation of nociceptive 
information transmission via spinal gate mechanism but also rely on a top-down descending 
pain inhibitory mechanism. 
Additionally, several studies in healthy individuals have found a positive association 
between EIH and the magnitude of conditioned pain modulation (CPM) (i.e. the paradigm 
of pain inhibits pain) (35) which is known for evaluating the efficiency of descending pain 
pathways inhibition, involving both serotonergic and opioidergic mechanisms (5). 

However, Ellingson et al. 2014 (36) showed that CPM is only a minor contributor to EIH 
due to differences in temporal and spatial manifestations. In fact, a significant CPM effect 
was observed only during evoked pain procedures (i.e. pressure), whereas EIH occurred 
following exercise. Also, during CPM a significantly larger effect on pressure thresholds 
was found at the remote sites compared to local sites, whereas EIH was significantly larger 
in the exercising body part compared to remote sites (13). Therefore, local and segmental 
mechanisms appear to play important roles for EIH.  
Thus, the influence of exercise on reducing peripheral nociceptive sensitivity has 
also been explored as a mechanism of EIH (18). Preliminary data has provided indirect 
evidence that the hypoalgesic effect induced by exercise could be possible related to the 
modulation 
of 
the 
signal 
transmission 
via 
A-delta 
and 
C 
fibers. 
Using 
electroencephalographic evoked potentials, Jones et al. (2016) (11) verify that 
somatosensory-evoked potentials (SEPs) to noxious electrical stimuli delivered to the index 
finger were unchanged after elbow flexion isometric exercise at 40% MVC for 3 min, 
whereas laser-evoked potentials (LEPs) to noxious heat delivered to hand dorsum decreased 
after exercise. This result suggests that the peripheral nociceptor might be involved in EIH 
in humans, because laser heat stimuli activate peripheral afferents and electrical stimuli 
bypass the receptors by activating the axons of the nociceptive afferents (11). This was 
further corroborated by Jones et al. (2017) (37) where by blocking blood flow to an 
unexercised limb during cycling exercise, resulting in unchanged pressure pain threshold 
afterwards, provided evidence that factors that are released into the blood during exercise 
(opioids, cannabinoids, catecholamines), which interact with specific receptors, are able to 
inhibit the nociceptive signal and therefore modify input before reaching the central nervous 
system. Nevertheless, the effect of acute exercise on LEPs amplitude attenuation observed 
by Jones et al., (2016) (11) was not statistical different from the passive rest control 
condition. The approximately 230ms onset latency of LEP was only compatible with 
conductive velocity of fast myelinated A delta fibers. The augmented pressure pain 
threshold verified after exercise resulted from an assessment obtained over a period of 

seconds eliciting a sensation of gradually increasing pain, only compatible with the activity 
of non-myelinated C fibers, which were not directly measured by EEG. Thus, if exercise 
has greater effects on C compared to A delta fibers further investigation should be addressed 
on the desensitization role of both afferent fibers post exercise. 
 
On the other hand, the effects of long-term training on nociception have been poorly 
investigated (18). To our knowledge, only three studies examined nociceptive modulation 
after exercise training in healthy individuals. Jones et al. (2014) (38) observed increases in 
ischemic pain tolerance in non-exercising region (upper limb), but not in pain threshold, 
after cycling training for 30 minutes at 75% of VO2max 3 times/week for 6 weeks. Recently, 
in an attempt to establish intensity-dependent effects, O’Leary et al. (2017) (39) observed 
that 3 times/week for 6 weeks of high intensity cycling interval training but not volume-
matched moderate intensity continuous training increased ischemic pain tolerance in the 
upper limb. On the opposite, Hakansson et al. (2018) (40), in pain-free overweight 
individuals, only observed augmented pressure pain threshold over the exercised muscles 
after 6 weeks of moderate-intensity continuous cycling training and not after volume-
matched high intensity training. 
It is important to mention that none of the above studies tried to establish 
mechanisms underneath training-induced analgesic effects. Some reports indicate that 
exercise training decreases cardiovascular sympathetic modulation mainly via central 
command (41). However, it has also been proposed that training modulates the exercise 
pressor reflex at cardiovascular level by attenuating the peripheric discharge of group III 
and IV muscle afferents in response to noxious, metabolic and mechanical stimuli, and thus 
regulating the sensation of nociception. Sinoway et al. (1996) (42) reported that 4 weeks of 
forearm training reduce sympathetic responses and mean arterial pressure during rhythmic 
voluntary handgrip. In addition, there is compelling evidence that endurance-trained 
individuals have a lower exercise pressor response compared to untrained people (43, 44). 
Thus, the available data indicate some level of adaptation within the exercise pressor reflex 
gain following low-resistance high-repetition muscle training, and this is in accordance with 

the findings of Somers et al. (1992) (45).  
 
1.2 Analgesia and Performance 
The mechanisms underlying neural changes in response to specific experimental 
interventions can be investigated by measuring reflex responses, namely the H reflex and 
V wave (46). Despite both responses are under the influence of common neural 
mechanisms, H-reflex is more sensitive to changes in the intrinsic motoneuron properties 
and presynaptic inhibition (46, 47, 48), while the V-wave is more sensitive to 
modifications in supraspinal input to the motoneuron pool (49, 50). In addition, they are 
task and training dependent (51, 52, 53). Longitudinal studies have revealed positive 
changes in H-reflex after endurance (52) and Wingate-based training (53), which reflects 
improvements in spinal excitability. Resistance training has been shown to increase V- 
wave amplitude (51, 52) which is representative of increased supra-spinal neural drive. 
Recent studies suggest that fatigue development may be related, at least in part, with 
nociception (54), and it may play a role in exercise tolerance (1). During exercise, ascending 
group III and IV muscle afferents discharge in response to noxious, metabolic and 
mechanical stimuli within the contracting skeletal muscles to regulate nociception, muscle 
activation and peripheral fatigue development (55, 56). As exercise-induced nociception is 
present in nearly all forms of time-dependent competition, the requirement for 
understanding its influence on performance and how athletes respond to its presence is 
imperative (1). Thus, the ability of an athlete to tolerate exercise-induced nociception is a 
critical factor in successful sports performance (54). If neuromuscular signals are 
consciously interpreted as nociceptive, leading to adjustments in pace or intensity, then 
individuals with heightened pain tolerance may be able to generate higher levels of muscle 
power (1). If nociception acts as a moderator of “allowed” exercise intensity, then the 
mechanisms that affect the level of pain tolerance may theoretically enhance performance 
by enabling an athlete to exercise beyond the normal protective threshold (eliciting a greater 
metabolic stress) (57).  

The most common methods to examine the effects of group III and IV afferents is 
to maintain ischemia after a fatiguing contraction or trough intramuscular injection of a 
hypertonic saline solution (58). Recently, Norbury et al. (2021) (59) observed that induced 
muscle pain by hypertonic saline solution injection reduces endurance exercise performance 
through the exacerbation of neuromuscular fatigue, which origins in inhibitory feedback 
from group III/IV nociceptors, reducing central motor output. Muscle afferents adjust the 
motoneuronal responses induced by the stimulation at the cervicomedullary level 
(corticospinal pathway) during exhaustive contraction. Such motoneuronal activation might 
be affected rather by changes in the corticospinal excitability, altering the effectiveness of 
the motor unit recruitment as indicated by the amplitude of cervicomedullary motor evoked 
potentials (CMEPs) or thoracic motor evoked potentials (TMEPs) measured through 
transcranial magnetic stimulation (TMS) (60). For the upper-limbs, Martin et al., (2006) 
(61) verify that after 2-min MVC of the homonymous or antagonists muscles the decrease 
of the CMEPs on elbow extensors was maintained post contraction by holding the muscle 
ischemic with a cuff inflated, and this was attributed to the maintained discharge of group 
III and IV afferents. Despite this, the same effect was not observed for the elbow flexors 
muscles (62, 61). For the lower-limbs, surprisingly, Kennedy et al., (2016) (63) showed that 
maintaining fire of group III and IV afferents with ischemia post 2 min MVC of the 
homonymous or antagonists’ muscles had no effect on knee extensors TMEPs. Unlike 
CMPEs or TMEPs pathway, H-reflex, apart from motoneuronal excitability, is also 
influenced and allows to assess pre and pos synaptic inhibition mechanisms (60). It was 
observed that H-reflex amplitude diminishes during sustained submaximal contractions at 
25% and 50% MVC of the abductor pollicis brevis muscle (64). Also, H-reflex amplitude 
evoked in the wrist flexor muscles is reduced by metabosensitive group III and IV afferents 
activation, trough hypertonic saline solution injection, suggesting that the spinal motor 
reflex activity seems to be uniformly affected by inputs from these afferents (65). Not only 
the evidence is somewhat conflicting regarding the TMS research, it is important to note 
that in the H-reflex above-mentioned studies (64, 65), the effects of induced muscle fatigue 

or pain were explored without constructing full recruitment curves individually at each time 
point. Instead, H reflex was obtained at single current intensities compatible with a given 
%Mmax. Interpretations based on this approach are seriously limited, because it does not 
allow determining whether the pre-selected current intensity induces a response falling in 
the ascending part of the recruitment curve, relevant for motoneuronal sensibility 
assessment. 
Furthermore, to our knowledge, research that reported increases in nociception 
tolerance after long-term training also observed improvements in performance at post-
training time point (i.e. VO2max) (38). Unfortunately, the relationship between improved 
exercise performance and EIH was not explored. Moreover, since training modulates the 
amplitudes of evoked H and V-wave potentials (51, 52, 53), it is important to examine 
whether the interaction between EHI and enhanced exercise performance originates at the 
spinal or supraspinal level. Ultimately, the training-induced analgesic effect might enhance 
H-reflex and V-wave excitability, leading to an improvement in motor performance.  
1.3 Purpose 
Since pre and pos synaptic inhibition of group Ia afferents can both be affected by 
metaboreflex activation (66), explaining H-reflex inhibition under these conditions, further 
research is needed to explore whether analgesic effects of post-acute and chronic exercise 
are partially dependent on lower H-reflex inhibition by group III and IV muscle afferents 
desensitization. In addition, it is important to note that the available research examining 
the interaction between exercise (acute and chronic) and nociception did not compare the 
effect of different exercise modalities. Since discharge rate of III and IV afferents is highly 
dependent on specific task-metabolic requirements (67), it is relevant to determine if 
exercise modality differentially modulates the possible analgesic response via 
metaboreflex desensitization (acute and chronic desensitization).  
Therefore, with this project, we aim at exploring the influence of experimentally 
induced metaboreflex activation (group III and IV afferents) (maintained submaximal 

isometric contraction followed by blood-flow occlusion) Vs. a control condition (no 
isometric contraction nor blood flow occlusion), on H-reflex recruitment curve and V wave 
excitability (aim #1). Since the activation of group III/IV afferents can inhibit the 
corticospinal pathway (61) we hypothesize that the normalized maximal amplitude and the 
slope of the ascending limb of the H-recruitment curve will decrease with metaboreflex 
activation. Additionally, we intend to examine the acute impact of different exercise 
modalities (resistance, supramaximal and endurance) on nociception sensibility (by the 
application of a cold tolerance test and by determining pressure pain threshold) (aim #2). 
This will allow us to investigate the role of different acute exercise paradigms on possible 
metaboreflex desensitization in its relationship with altered neuromuscular responses at 
post-exercise time point (attenuated inhibition of H-reflex and V-wave amplitude during 
metaboreflex activation). Also, once past research has shown that muscle metaboreflex 
activation reduces the sensitivity of the baroreflex (68), we will measure arterial pressure 
(AP) and heart rate variability (HRV) during cold pain tolerance testing. Despite all 
exercise modalities could enhance reducing pain sensibility after exercise, we hypothesize 
that resistance and supramaximal exercise stimuli will enhance nociceptive tolerance via 
metaboreflex desensitization compared to that seen with endurance exercise (attenuated 
increase of AP and sympathetic component of HRV during cold pressor test).  Finally, we 
intend to explore the impact of long-term resistance, supramaximal and endurance training 
on nociceptive sensibility, using the same tests of aim #2 (aim #3). We hypothesize that 
chronic EIH will be of greater magnitude after supramaximal and resistance exercise 
training vs. endurance training. In addition, we hypothesize that decreased nociception will 
be related with attenuated inhibition of H-reflex and V-wave amplitude and attenuated 
increase of AP and sympathetic component of HRV during metaboreflex activation after 
training and this will result in increased exercise performance via heightened H-reflex and 
V-wave excitability. 

2. Research Plan and Methods 
 
2.1 Aim #1 
 
2.1.1 Participants 
 
We will recruit a total of 16 participants ((G Power 3.0.10 software) to achieve 
80% of correctly reject the null hypothesis, based on Duchateau et al., (2002) (64)). 
Specific inclusion and exclusion criteria are discussed in subsection 2.4.1 
2.1.2 Study design 
To study the influence of experimentally induced metaboreflex activation on H-
reflex and V-wave excitability, participants will be tested on 2 different conditions 
(with and without maintained submaximal isometric contraction followed by blood 
flow occlusion, using a randomized, crossover approach). The experimental design is 
illustrated in figure 1. 
2.2 Aim #2 
2.2.1 Participants 
The intervention study will enroll 24 participants ((G Power 3.0.10 software) to 
achieve 80% of correctly rejecting the null hypothesis, based on Jones et al., (2016) 
(11)). Eight participants will be randomly allocated to each exercise modality (i.e. 
resistance, supramaximal and endurance). Specific inclusion and exclusion criteria are 
discussed in subsection 2.4.1. 
2.2.2 Study design 
In a randomized and crossover trial, on 2 different days, participants will be 
tested before and after performing acute bouts of different exercise modalities or 

passive rest. Participants will be tested for: (1) pressure pain threshold, (2) cold pain 
tolerance with concomitant measures of AP and HRV, (3) the influence of metaboreflex 
activation (maintained submaximal isometric contraction followed by blood flow 
occlusion) on H-reflex and V-wave excitability. For those enrolled in endurance and 
resistance exercise modalities, participants will visit the laboratory on a previous day 
for VO2max and maximal repetition assessments for rigorous exercise prescription on 
experimental day. The experimental design is illustrated in figure 2. 
2.2.3 Exercise protocols 
Participants allocated to resistance exercise will perform 3 sets of 2 bilateral leg 
exercises (leg press and seated calf raise). The load intensity will be set at 8-12 maximal 
repetitions. Participants allocated to endurance exercise will perform 30 min on the 
speed compatible with 75% of VO2max. Participants allocated to supramaximal exercise 
will perform 2 bouts of 30 s of supramaximal cycling exercise, using a relative load 
equivalent to 7.5% of the individual body mass (Monark Ergomedic 894E®). Both bouts 
of supramaximal exercise will be interspersed with 4 min of active recovery. 
 
2.3 Aim #3 
2.3.1 Participants 
We will recruit a total of 30 participants. Sample size is overestimated in order to 
allow us to accommodate an attrition rate of ~ 20% during follow up, once participants 
will only be eligible for final testing if they attain at least 85% of the training sessions, 
with the goal of allocating at least 8 participants to each of the three training groups (G 
Power 3.0.10 software; to achieve 80% of correctly reject the null hypothesis, based on 
Jones et al. 2014 (38)). Specific inclusion and exclusion criteria are discussed in 

subsection 2.4.1 
2.3.2 Study design 
Each intervention group will perform 6 weeks of exercise training (resistance, 
supramaximal and endurance training). Participants allocated to each training group 
will be evaluated before and after training. On one day participants will be tested for 
pressure pain threshold and cold pain tolerance with concomitant measures of AP and 
HRV. The influence of metaboreflex activation (maintained submaximal isometric 
contraction followed by blood flow occlusion) on H-reflex and V-wave parameters will 
also be measured. On another day, participants will be evaluated for resting AP and 
HRV, neuromuscular adaptations and performance. The experimental design is 
illustrated in figure 3. 
2.3.3 Training protocols 
All participants allocated to each training group will exercise 3 days/week at 
the same time of the day, with 24 h of interval between sessions. Those enrolled in 
resistance training will perform 3 sets of 2 bilateral leg exercises (leg press and seated 
calf raise). In the first week, the load intensity will be set at 15-18 maximal repetitions. 
From then on, the load intensity will be performed over 8–12 maximal repetitions. 
The supramaximal training regimen will consist of 30 s of supramaximal cycling 
exercise (Monark Ergomedic 894E®), interspersed with 4 min of active recovery, using 
a relative load equivalent to 7.5% of the individual body mass. During the first four 
weeks of training, participants will complete two 30s all-out sprints per session. From 
then on, volume will be increased to four 30s all-out sprints per session. 
Endurance training will be performed on a motorized treadmill (Jaeger®, 

Laufergotest, Germany). During the first week of training, relative intensity will be set 
at 65-75% VO2max and each training session will last 30–40 min. From then on, the 
duration of the training sessions will be increased to 40–50 min and the treadmill speed 
will be set to elicit 75–85% VO2max  
2.3.4 Exercise Performance 
2.3.4.1 Resistance training group 
Both before as well as after training, participants will be evaluated for maximal 
and submaximal knee extension strength. Measurements will be taken in the seated 
position, with the participants maintaining their knee flexed at 70º (69). MVC will be 
determined based on the highest peak torque obtained in 3 trials, interspersed by 60 s 
of passive recovery. Then, participants will be asked to maintain 40% of MVC until 
failure. Visual feedback from the dynamometer software will be provided. Task 
termination will occur whenever the participants exhibit strength decrements ≥ 10% for 
more than 10 s.  
2.3.4.2 Endurance training group 
 
Exercise performance will be determined based on the maximal distance 
completed by each participant in response to a time-trial run. For the time-trial run, 
both before and after training, participants will be asked to endure a submaximal 
treadmill running for as long as possible at the speed compatible with 50% of the 
distance between the 1st ventilatory threshold and VO2max (∆50 speed). 
Before each time trial, on a different day, participants will complete a treadmill 
ramp protocol to volitional exhaustion. This test will be used with two specific 
purposes: 1) to determine participants VO2max, 1st ventilatory threshold, and the ∆50 

speed; 2) to prescribe the treadmill running speeds for the training period.  
2.3.4.3 Supramaximal training group 
 
Both before as well as after training, participants will perform the maximal 
number of 30 s maximal all-out cycling trials (70), interspersed by a 4-minute of active 
recovery period, until mean power value decreases to 50% of the first supramaximal 
bout. 
2.4 Common methodology 
2.4.1 Participants (aim #1, #2 and #3) 
 
Recreationally active men aged 18-30 years will be recruited. Women will not 
be included once responses to experimental pain have been demonstrated to vary 
according to the menstrual cycle phase (71). Participants will be recruited from the 
local communities and from the Faculty surroundings via word-of-mouth for this study. 
Exclusion criteria will include known metabolic, cardiovascular, respiratory, 
orthopedic disease and taking medication for the treatment of pain or having any pain 
related conditions. Each participant will complete a health- screening as well as a 
physical activity questionnaire and baseline measures (body mass, height and blood 
pressure). Finally, participants will be provided with verbal information about the 
requirements and demands of the study and required to give written, informed consent 
to the experimental procedures, which was approved by the Faculty of Human Kinetics 
ethics committee (CEFMH Nº33/2021) and according with the Declaration of Helsinki. 
Participants will be told to avoid strenuous physical activity 24 hr, caffeine ingestion 4 
hr and analgesics intake 6 hr prior to each testing session (59). 
2.4.2 MVC (aim #1, #2 and #3) 
 

All neuromuscular assessments will be made on the participants’ dominant lower 
limb (plantar-flexors), using a Biodex System 3 Pro isokinetic dynamometer (Biodex 
Medical System 3). All testing sessions will be conducted between 07:00 and 11:00 
hours, to avoid daily variations in muscle strength related to human circadian rhythms. 
Measurements will be taken in the seated position, with the participants maintaining their 
hips and knee flexed at 120° and ankle at 110° of plantar flexion (70). The flexed position 
at the knee joint will be used to reduce the mechanical contribution of the gastrocnemii 
to the plantar-flexor torque. Participants will be asked not to alter their posture and to 
focus on the task. MVC and rate of torque development (RTD) will be determined for 
plantar flexor muscles, based on three isometric contractions, lasting 5 s each. A 60 s 
interval of rest will be respected between trials. Participants will be instructed to exert 
their maximum force as fast and hard as possible with verbal encouragement and visual 
feedback from the dynamometer software being provided. The highest peak torque value 
will be defined as the participants MVC value (72, 73). 
2.4.3 Surface EMG (aim #1, #2 and #3) 
 
Surface EMG will be recorded continuously from EMG electrodes placed on 
soleus and anterior tibial muscles (Delsys DE 2.x series EMG sensors). After skin 
preparation, a 2-slot adhesive interface will be applied on the EMG sensor to firmly 
stick the sensor to the skin. A conductive reference electrode will be placed around the 
left ankle of each participant (36). Surface EMG signals will be band-pass filtered (10 
– 2000 Hz) and amplified using a Delsys Bagnoli-8 amplifier to a total gain of 1000. 
Then, a 16-bit analog-to-digital converter (National Instruments, USB-6251) will be 
used to sample the signal at 10 kHz. EMG data will be recorded in synchrony with the 
torque signal originating from the Biodex System, using Mr. Kick software (Knud 

Larsen, SMI, Aalborg University) (73). 
2.4.4 Percutaneous Electrical Nerve Stimulation (aim #1, #2 and #3) 
 
A constant-current isolated stimulator (STIMSOLA, Biopac Systems; Inc; CA, 
US) will be used to stimulate posterior tibial nerve. The cathode electrode (8 mm 
diameter, Ag-AgCl) will be placed on the popliteal fossa and the anode (5 x 10 Compex, 
Medical SA) proximal to the patella. Each participant will receive a range of electrical 
stimuli (1-40 mA) to determine the position eliciting the greatest response with the 
minimum stimulus intensity. The optimal position will be identified using a handheld 
cathode ball electrode. Finally, stimulation electrode will be fixed with rigid straps and 
taping (72, 73). 
2.4.5 Standard H-recruitment curves (aim#3) 
Before and after long-term training, standard methods will be used for M-wave 
and H-reflex recordings (52). These measurements will be taken while each participant 
maintains a submaximal low intensity contraction of the plantar flexors (10% MVC) 
with visual feedback from the dynamometer software being provided. The upper 
current intensity will be determined, firstly, by progressively increasing the current 
intensity by 5 mA from 0 until there is no further increase in peak torque or in peak-to-
peak M-wave amplitudes. At each intensity level, three stimuli will be delivered at 3-
seconds intervals. To construct the H-recruitment curves, the upper current intensity 
(compatible with maximal M-wave) will be divided into 22 segments equally separated 
on a logarithmic scale, with 16 stimuli/intensity randomly applied to construct the 
recruitment curves, delivered at 3s intervals. 
2.4.6 V wave recordings, contractile properties and octet contractions (aim #3) 
Before and after long-term training, V waves will be elicited during 5 MVCs, with 1 

min of rest between trials, via supramaximal electrical stimulation (150% of the current 
needed to evoke maximal M wave; 1-ms single square pulse) delivered to the tibial 
nerve when torque exceeds 90% MVC (52). Contractile muscular properties will be 
measured from muscle twitches evoked by three single pulses (1-ms square pulse) at 
120% of the current needed to evoke maximal M-wave, delivered every 12s (74). 
Evoked octet contractions (8 pulses at 300Hz), which has been found to elicit the 
maximum capacity of rate of torque development, will be applied through three 
supramaximal pulses (at 120% of the current needed to evoke maximal M-wave), 
delivered every 12s (74). 
2.4.7 H-recruitment curves and V-wave recordings with metaboreflex activation (aim 
#1, #2 and #3) 
During the blood-flow occlusion trials, to ensure metabolite accumulation 
participants will previously perform a plantar-flexion contraction during 3 minutes at 
40% MVC (aim #1). 25% MVC will be used in aim #2 and #3, in order to avoid any 
potential hypoalgesic effect to interfere with the analgesic effect of exercise or training 
protocols. There is evidence that 25% MVC isometric contraction for 5 min doesn’t 
produce elevations in pain threshold (29) at the same time it is a contraction with 
sufficient intensity to produce nociceptive afferents activation (64). Then, using a rapid 
inflator system (Hokanson E20 AG101, Bellevue, WA), an adult-size cuff 12 x 124 cm 
(Hokanson, SC12L, Bellevue, USA) (placed on the upper portion of the thigh) will be 
inflated to a pressure of 300 mmHg to ensure complete blood-flow occlusion (75). This 
procedure will preserve the activation of group III/IV afferent fibers for subsequent 
measurement of H-reflex and V-wave excitability. To construct the H-recruitment 
curves, the upper current intensity (compatible with maximal M-wave) will be divided 

into 22 segments equally separated on a logarithmic scale, with 4 stimuli/intensity, to 
limit the time under ischemia (~4 min), randomly applied to construct the recruitment 
curves, delivered at 3s intervals. After 2 min rest, the isometric plantar flexion 
contraction will be repeated with subsequent thigh circulatory occlusion. V waves will 
be elicited during 5 MVCs with 15 s of rest between trials (63) to limit the time under 
ischemia (~2 min), via supramaximal electrical stimulation (150% of the current 
needed to evoke maximal M wave; 1-ms single square pulse) delivered to the tibial 
nerve when torque exceeds 90% MVC. In control condition of aim #1 this H-reflex and 
V-wave collecting procedure will also be used but without sustained isometric 
contraction followed by blood flow occlusion. In aim #2 it will be assessed 15-30 min 
post-exercise or rest. 
2.4.7.1 Ischemia pain ratings (aim #1, #2 and #3) 
 The participants will be asked to rate the intensity of ischemia pain sensation 
using a 11-point (0−10) numerical scale (76), every 60s. The left end of the scale (=0) 
will be anchored with no pain, the middle of the scale will be anchored with moderate 
pain (=5) and the right end of the scale will be anchored with worst pain possible (=10). 
2.4.8 Pressure pain threshold (aim #2 and #3) 
 
Pressure pain threshold (PPT) will be assessed on the right rectus femoris 
(exercising muscle) and biceps brachii (non-exercising muscle). We will use a handheld 
algometer to perpendicularly deliver pressures to the participants’ skin, and the pressure 
force will be increased at a rate of approximately 1 kg·s−1. The pressure will be stopped 
when they feel the pressure became painful. This procedure will be repeated three times 
for each test site. To minimize the impacts of repeated stimuli on the experimental 
outcomes, a recovery period of 20–30 s will be set between each measurement. The pain 

pressure threshold will be recorded as the average of these three measures (34). In aim 
#2 it will be assessed 0-15 min post-exercise or rest. 
2.4.9 Cold pain tolerance (aim #2 and #3) 
 
A cold pain tolerance (CPT) test, with the participants comfortably seated while 
immersing the right foot into a tank 5cm above the ankle joint (13) in cold water at ~2ºC 
(1º - 4º) (77) will be used to determine participants pain tolerance before and after both 
acute exercise and long-term training. Water temperature will be adjusted through 
immersion of ice until achieving the desired temperature. Participants will be instructed 
to remove their foot from the water at any time when unable to support strong cold 
nociception sensation. In aim #2 it will be assessed 0-15 min post-exercise or rest (after 
pressure pain threshold assessment). Once breathing frequency might modify the 
subjective level of cold pressor pain (77), we will fix ventilatory frequency using a 
metronome for standardize the duration of the inspiration and expiration phases (2s 
each). 
2.4.9.1 Cold pain ratings (aim #2 and #3) 
The participants will be asked to mark the intensity of cold pain sensation using a 
visual analogue scale (VAS) with a horizontal line of 100mm (78), every 30 s. 
2.4.10 Arterial pressure and heart rate variability (aim #1, #2 and #3) 
AP will be assessed using an automatic monitor (Tango SunTech Medical 
Morrisville, NC) during thigh blood flow occlusion procedures to confirm the sustained 
activation of the metaboreflex. In addition, AP will be measured before and after acute 
exercise and long-term training during cold pain tolerance testing (every minute). 
Cardiac autonomic regulation, both before and after acute exercise and long-term 
training during cold pain tolerance testing, will be assessed continuously from a Polar 

R-R recorder (Polar RS 800 CX, Finland) trough spectral decomposition of beat-by-beat 
HRV (79). Resting AP and HRV (continuous recording during 5 min) will also be 
assessed before and after training. 
2.4.11 Negative risk involved in the experiments and control countermeasures (aim #1, 
#2 and #3) 
The risk of injury during the course of the study are minimal. Nevertheless, 
participants will be asked to report any adverse reaction. Additionally, besides 
nociception, no past reports indicate complications derived from temporary total blood-
flow occlusion (75) nor cold-water limb immersion (77). The application of electrical 
current to the tibial nerve can generate some discomfort as well as the Wingate testing 
and the training protocols. Participants will be monitored and supervised during each 
experimental session. No insurance is foreseen for this study. 
2.4.12 Data analysis (aim #1, #2 and #3) 
 
Neuromuscular function The peak-to-peak amplitude of the M and V waves will 
be computed offline from unrectified EMG epochs. Although H waves will be normalized 
to the corresponding Mmax, V waves will be normalized to the supramaximal M wave 
(Msup) elicited concomitantly with the V wave (V/Msup) (52). V waves with concomitant 
Msup < 90% of the highest Msup will be excluded from further analyses to ensure stable 
conditions (73). A general squares model will be fitted to the ascending part of the H-
reflex recruitment curve and the following parameters will be then extracted (80): (a) 
current intensity at H-reflex threshold, (b) current intensity at 50% of Hmax, (c) current 
intensity at Hmax and (d) slope of the ascending limb of the recruitment curve at 50% of 
Hmax. Data analysis will be computed exclusively for recruitment curves with r square 
values >0.90 (80). The amplitude of background surface EMG will be derived from the 

average rectified value (ARV) analyzed over an epoch of 500 ms before stimulation. The 
ARV values will be normalized to those obtained during the highest MVC (ARV 
computed from a 500-ms time period centered at peak force) and expressed as a 
percentage. During the MVCs performed with supramaximal stimulation, EMG ARV will 
be computed using a 500-ms period before stimulation. Then, these values will be 
normalized to the corresponding M wave obtained during supramaximal stimulation (52). 
Analyses of twitch contractions will be based on the average of the 3 supramaximal 
measurements. We will determine maximal twitch torque amplitude, time to peak twitch 
torque and half-relaxation time (74). Volitional and evoked RTD will be measured in 
several time windows (0-50ms, 50-100ms and 100-150ms) from force onset, using the 
trial with the highest peak RTD value (74). 
Heart rate variability (aim #2 and #3) Power spectral analysis will be performed 
based on autoregressive modeling, following data detrending and re-sampling (4Hz) 
(Kubios HRV Analysis Software for Windows, University of Kuopio, Finland). Two 
main components will be considered: low frequency (LF) – frequency band from 0.04 to 
0.15 Hz; high frequency (HF) – frequency band from 0.15 to 0.4 Hz. The ratio LF/HF 
will be calculated as an index of sympathovagal balance (79). 
2.4.13 Statistical analysis (aim #1, #2 and #3) 
 
Normality will be verified through the Shapiro-Wilk test. Univariate ANOVAs 
will be used to explore differences in the impact of blood-flow occlusion vs. non-blood 
flow occlusion on each H-reflex and V-wave variable (aim #1). Repeated measures 
ANOVA will be computed to determine significant main effects for time (pre- vs. post-
acute exercise), condition (resistance vs. supramaximal vs. endurance exercise) and 
time-by-condition interactions on pain tolerance/threshold/ratings, AP, HRV and H-

reflex parameters and V-wave amplitude with metaboreflex activation (aim #2). 
Repeated measures ANOVA will be computed to determine significant main effects 
for time (pre- vs. post training), condition (resistance vs. supramaximal vs. endurance 
training) and time-by-condition interactions on exercise performance, pain 
tolerance/threshold/ratings, AP, HRV, H-reflex parameters and V-wave amplitude 
with metaboreflex activation, and neuromuscular adaptations to training (aim #3).  
Correlations 
between 
the 
impact 
of 
exercise 
or 
training 
on 
pain 
tolerance/threshold/ratings and performance on H-reflex, V-wave, AP and HRV 
variables will also be explored. Significance level will be set at p < 0.05. All data will 
be treated using SPSS 25.0 for statistical analysis. 
2.4.14 Final study procedures (aim #1, #2 and #3) 
 
All data will be compiled in Neuromuscular Research Laboratory private cloud. 
We will ensure the participants confidentiality by attributing a code number to each 
person. Only the main researcher and his supervisor will have access to the data, 
and always through the code numbers. All participants will have access to their results 
via an individual report. 
2.4.15 Conflicts of Interest 
 
There are no conflicts of interest to declare 

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Figure 1. Experimental design of aim #1 

 
 
 
Figure 2. Experimental design of aim #2 
 
 
 
 
 
 
 
    
    
        
               
                
       
        
            
           
           
      
             
         
             
      
           
                      
   
   
  
  
   
   
                                       
                                       
                            
                             
            
            
   
                                       
   
   
                                       
   
            
    
   
   
   
   

 
 
Figure 3. Experimental design of aim #3