Participants

Thirteen healthy male volunteers (mean ± SD: age 31 ± 7 years, height 1.76 ± 0.08 m, body mass 75 ± 11 kg) provided written, informed consent to participate in the present study, which was approved by the Ethics Committee of Sport and Health Sciences (University of Exeter). After being informed of the experimental procedures and associated risks, all participants completed a medical health questionnaire to ensure they could safely consume ACT prior to performing exhaustive exercise. Subjects were not consumers of any ‘pain relief’ medication (prescription or non-prescription) over the course of the study. None of the subjects had a history of motor or neurological disorders.

Experimental design

Subjects visited the laboratory on three occasions over a 3- to 4-week period with all tests separated by at least 1 week and conducted at a similar time of day (± 90 min) to limit changes in quadriceps strength and to account for diurnal variations in neuromuscular excitability. The first laboratory visit was used to familiarise subjects to the measurements and experimental protocol described below. During these sessions, the settings and placement of the dynamometer and EMG and peripheral nerve stimulation electrodes were recorded for each subject. Subsequently, subjects performed the fatiguing protocol under two conditions (see ‘Experimental protocol’): placebo (PL) and ACT.

Experimental protocol

Subjects completed a preliminary trial for familiarisation to the measurement techniques and experimental protocol. An isokinetic dynamometer (Biodex System 3, Shirley, NY, USA) was used in all tests and adjusted so that the axis of rotation of the lever arm was in line with the lateral epicondyle of the right femur. Subjects were seated with the hip and knee joints at relative angles of 155° and 90°, respectively. The remainder of the chair settings were recorded and replicated in all subsequent trials to ensure an identical body position was assumed throughout the experimental trials. The semi-supine position was employed as it permitted better access to the superficial femoral nerve for peripheral nerve stimulation as verified during pilot testing. Inelastic padded Velcro straps were fastened at the ankle, quadriceps, hip and shoulders to maintain a stable body position. The procedures adopted during the familiarisation trial were replicated in all experimental trials.

The trials on visits 2 and 3 were completed in a double-blind, randomised fashion using a cross-over experimental design. Prior to each visit, subjects were required to refrain from caffeine (for at least 12 h), strenuous exercise and alcohol (for at least 24 h), analgesics and any form of anti-inflammatory drug (for the duration of the experimental trial) and to arrive in a fully rested and hydrated state. Subjects were instructed to maintain their usual diet and exercise regime during the study. 1 g of maltodextrin (placebo) or 1 g of acetaminophen was ingested orally, 60 min prior to the exercise bout such that the start of the exercise trial was expected to coincide with attainment of the peak plasma [acetaminophen] (Anderson et al. 2008; Forrest et al. 1982). Following oral ingestion, ACT is rapidly absorbed from the gastrointestinal tract and its bioavailability ranges from 70 to 90% (Forrest et al. 1982). The trials started with a standardised isometric warm-up routine (10 isometric contractions for 3 s at 50% of pre-exercise MVC as determined during familiarisation testing) and testing of the optimal EMG electrode, anode and cathode placement and stimulation intensity for peripheral nerve stimulation. Neuromuscular function was assessed pre-trial, during and (< 10 s) post-trial. Single peripheral nerve stimulation pulses were manually triggered at rest to determine pre-exercise neuromuscular function, namely the characteristics of the M-wave response (M-wave amplitude; M max ) to supramaximal nerve stimulation, contractility of the muscle (i.e., maximal rate of force development, half-relaxation time and contraction time), voluntary activation (VA) and potentiated twitch force (pTw). During MVCs, peripheral nerve stimulation pulses were triggered to occur as soon as a peak torque was achieved (typically 1.5 s into a 3 s contraction), each separated by a 40 s rest. The stimuli were also delivered 1–2 s after the cessation of the contraction to provide a resting twitch (rTw). Identical measurements were repeated as soon as possible (< 10 s) after the fatiguing exercise to determine post-exercise neuromuscular function (see Fig. 1).

Fig. 1 Schematic of the procedures used prior to (panel a), during (panel b) and within 10 s following (panel c) the 60 maximal isometric voluntary contraction (MVC) protocol. 10 s separated each single pulse stimulation administered at rest (small dashed arrows). a (pre) c (post) 45 s rest period separated maximal efforts (MVCs). Single pulse stimuli were administered during peak force production of MVCs (large solid arrow) and immediately (1–2 s post; small grey arrows). b 60 MVC protocol of the knee extensors. The figure presents a period of 30 s which is repeated sequentially for 5 min. Each MVC was held for 3 s and interspersed by a 2 s passive recovery period. Every 6th MVC was accompanied by single pulse stimuli administered during peak force production (large solid arrow) and immediately following (< 2 s post; small grey arrows). This cycle was repeated 10 times such that the protocol spanned 5 min requiring the completion of 60 MVCs. MVC, maximal voluntary isometric contraction. Surface electromyography (EMG) was measured throughout Full size image

The intermittent isometric contraction protocol used in this study had a similar design to that presented by Burnley (2009). The protocol consisted of repeated brief MVCs (3 s contraction, 2 s rest), timed via a visual prompt to ‘go’ and ‘relax’ (separate to the torque output screen), accompanied by the same verbal instructions from the experimenter. The protocol was terminated when the subjects completed the 60th MVC. Every 6th contraction was accompanied by peripheral nerve stimulation during and post-MVC (as described for pre- and post-trial measurements). Subjects were not given a visual representation of the torque produced during each MVC or made aware of the number of MVCs elapsed during the protocol. Subjects were instructed to continue to perform maximal contractions throughout. The coefficient of variation for MVC performance (mean torque through 60 MVCs) and electromyography root mean square (EMG RMS ) amplitude using this experimental set up was 2.9% and 4.8%, respectively as calculated during pilot testing.

Measurements

Torque

Knee extensor torque from the Biodex isokinetic dynamometer was sampled at 1000 Hz and low-pass filtered at 40 Hz, before being displayed on a wide screen monitor using Spike2 (CED, Cambridge, UK). Torque was expressed throughout as a percentage (%) of pre-exercise MVC.

Electromyography

Surface EMG activity was recorded from vastus lateralis, vastus medialis and rectus femoris of the quadriceps and biceps femoris of the hamstring of the right leg using active bipolar bar electrodes in a single differential configuration (DE2.1, DelSys Inc, Boston, MA, USA). The electrodes were placed over the respective muscle bellies (SENIAM guidelines). Double-sided adhesive tape and a hypoallergenic medical tape were used to ensure the EMG sensor stability for recording electrodes. The skin area underneath each EMG electrode was shaved, then exfoliated and cleaned with alcohol to minimise the skin impedance. The EMG and torque signals were pre-amplified (1000×), band-pass filtered (20–450 Hz, Bagnoli-8, DelSys Inc, Boston, MA, USA), and then transferred to a computer with a sampling frequency of 2 kHz and high-pass filtered at 10 Hz. EMG and torque data were recorded continuously and digitised synchronously with 16 bit resolution via an A/D converter (± 5 V range, CED 1401 power, Cambridge, UK). The electrodes were used to record: (1) evoked muscle action potential (peak-to-peak amplitude of the M-wave); and (2) EMG to estimate muscle activity and the output of spinal motoneurons (motor unit recruitment and firing frequency). EMG was average rectified using the root mean square method (EMG RMS ). EMG RMS was then normalised to the pre-exercise maximum (or maximal EMG signal) and the local M-wave amplitude (closest measure of the M-wave to the MVC) to exclude any changes to the EMG trace to changes in local excitability. The ground electrode was placed over the patella of the right leg.

Peripheral nerve stimulation

Electrical stimulation was applied by a constant current stimulator (Digitimer Stimulator DS7, Digitimer, UK). M-waves were elicited by supramaximal percutaneous electrical stimulation of the femoral nerve (200 µs duration). The cathode was placed over the femoral nerve in the inguinal fossa, approximately 3–5 cm below the inguinal ligament in the femoral triangle. Once the M-wave was elicited, the maximum amplitude (peak-to-peak) of the M-wave was determined (M max ) for the vastus lateralis and vastus medialis. To determine the stimulation intensity (current), single stimuli were delivered in 20 mA step-wise increments from 100 mA until a plateau in quadriceps rTw and M-wave were observed. To ensure a supramaximal response, the current was increased by an additional 30% (mean ± SD current = 194 ± 81 mA; Burke 2002; Goodall et al. 2010; Neyroud et al. 2014). The average M max was obtained from three stimuli, with ~ 8–10 s separating each pulse at rest. Peak torque, maximal rate of force development, half-relaxation time and contraction time were analysed for all rTw and pTw.

Data analyses

Data were analysed using a custom written script developed in Spike2 software (CED, Cambridge, UK). Mean torque for each 3 s contraction was determined for all tests as the mean value over a 1 s period which approximated the plateau level of the highest torque (i.e., 500 ms before and after the peak torque). The pTw was calculated as the peak torque achieved following the single pulse delivered 1 s post-MVC. The twitch torque superimposed onto the peak force production of the MVC (sTw) was calculated as the increment in torque immediately following the pulse during MVCs. The torque-impulse was calculated as the area under the torque-time curve by accumulating the time integral of each MVC (3 s) by the difference in torque between MVCs. The end-test torque (i.e., CT) during the 60 MVC test was defined as the mean of the last 12 contractions (i.e., the last 60 s; Burnley 2009). The W′ was calculated as the area above the CT from the torque-time curve (i.e., impulse above CT). Central fatigue was assessed as the maximal voluntary activation of the motoneuron pool (VA, %), calculated using the interpolated twitch method from peripheral nerve stimulation (Merton 1954). Specifically, the increment in torque evoked during the MVCs was expressed as a fraction of the amplitude of the potentiated twitch produced with the same stimulus in the relaxed muscle post-MVC. The level of voluntary drive was then quantified as a percentage: [1 − evoked torque (superimposed on voluntary torque, sTw)/ (potentiated twitch torque, pTw) × 100] (i.e., Allen at al. 1998).

The changes in maximal voluntary torque, rTw, pTw, VA and EMG RMS , were used to quantify peripheral fatigue and central fatigue. The maximal EMG was taken from the first MVC during the 60 MVC task and compared to the last MVC at task end. The neuromuscular parameters extracted from the three sets of maximal contractions completed post-exercise were tested for statistical differences between sets of contractions and then compared to the first set of MVCs completed pre-exercise (Froyd et al. 2013; Pageaux et al. 2015; Doyle-Baker et al. 2017). Neuromuscular function was also measured for each of the stimulated contractions during the exercise and normalised to the corresponding pre-exercise values at 100% MVC. All neuromuscular parameters and torque were averaged across the protocol using 6 (30 s) bin averages.

Statistics