Subjects aged ≥ 18-years referred to our hospital for the diagnosis of chronic fatigue were recruited between April 2018 and June 2019. Inclusion criteria included: at least 6 months of chronic fatigue and at least four symptoms of inability as required for the diagnosis of ME/CFS [1, 2]. Exclusion criteria were: a history of any other pathology confounding the ME/CFS diagnosis, including multiple sclerosis, auto-immune disease (lupus and sicca syndrome), diabetes, congestive heart disease, non-treated hypothyroidism, inflammatory muscle disease, or a significant psychiatric illness (e.g. major depression or psychosis) or pregnancy. Patients with drug abuse during the previous 12 months were also excluded.
The study protocol was approved by the French institutional review boards for human studies (ANSM and CPP Ouest II) and written consent was obtained from all patients to measure their maximal capacity at work.
Electromyography (EMG) recording and analysis
Bipolar Ag–AgCl surface electrodes (Medtronic, 13 L 20 Skovlunde, Denmark) were used to measure EMG voltage from the RF and FDL muscles on the dominant side of the body, as in previous studies [8,9,10,11,12,13]. The EMG signal was amplified (Nihon Kohden, Tokyo, Japan; common mode rejection ratio, 90 dB; input impedance, 100 mOhm; gain, 1000–5000) with a frequency band ranging from 10‒10,000 Hz. Compound muscle mass action potentials (M-waves) were evoked by direct muscle stimulation, using a constant-current neurostimulator (Grass, Quincy, MA, USA) delivering supramaximal shocks with 0.1 ms rectangular pulses through an isolation unit. Stimulating skin electrodes were fixed perpendicularly to the muscle axis from either side of the recording electrodes. An oscilloscope (model DSO 400; Gould, Ballainvilliers, France) was used to measure the average M-waves from eight successive potentials and to calculate the peak M-wave amplitude and its duration, and the conduction time, that is the time between the stimulus artefact and the peak.
Maximal handgrip strength
Maximal handgrip strength was also measured to determine the degree of force failure. Maximal handgrip strength was measured with the wrist in the neutral position with a pronated forearm to hold the handgrip device (model 5401; Takei Scientific Instruments Co Ltd, Niigata-City, Japan), as recommended by de Ponte et al. . Study participants were instructed to perform three maximal handgrips sustained for 3 s. The highest handgrip strength of three contractions, expressed in Newtons (N), was considered the maximum. Each forearm was tested. The reference values were those reported by Steiber .
Five ml of heparinized blood was taken from each subject to measure the biochemical variables. Potassium concentration was measured at rest and at the end of exercise to evaluate K+ outflow in relation to exercise. Two blood markers of oxidative stress (TBARS and ORP) were used. Plasma TBARS concentration was measured according to the method of Uchiyama and Mihara . Plasma ORP was measured using a combined platinum electrode with an Ag/AgCL reference and a potentiostat (ArrowDox; Lazar Research Laboratories, Los Angeles CA, USA). Electrode calibration was performed using 10 different molarities of HCl. Two to three measurements of each plasma sample, separated by a 1-h interval, were performed. Between each measurement, the ORP of pure water was recorded and used to determine the shift with time of the rest value provided by the electrode, which was eventually used to correct the ORP sample values. The results are the average of 2–3 replications with a standard deviation of < 5%.
All patients underwent an exercise session on a cycle ergometer up to their maximal work rate supported for a 1-min period, often limited by muscle fatigue. As in our previous studies [8,9,10,11, 13], the protocol consisted of: (i) a 2-min rest period, during which all variables were measured and venous blood samples collected; (ii) a 1-min 20-W work load period used to reach the 1 Hz cycling frequency; (iii) a work period; and (iv) a 10-min recovery period. During the work period, the load was increased by 20 W every 60 s until fatigue obliged the subject to stop the exercise. Peak oxygen uptake (VO2max) was measured at the maximal work rate (Wmax) as was the maximal increase in heart rate. M-waves were recorded at 20, 60, 100 W, and at the maximal work rate reached. The ergometer was then unloaded and the subject continued to cycle for a 2-min recovery period to facilitate venous blood return from the legs. During recovery, M-waves were recorded at 2, 5 and 10 min. During the exercise session and the post-exercise recovery period the right forearm remained totally relaxed, the hand being placed on the ergocycle handlebar without grasping.
Power analysis for determination of sample size was founded on an assumption of 95% confidence intervals [95% CI] and 80% power. The Holm-Sidak test was used for both pairwise comparisons and comparisons versus a control situation (rest) to determine the significance of changes in M-wave amplitude and duration. The Student’s t-test was used to assess intergroup differences in M-wave changes between resting values of TBARS and ORP, and maximal changes in M-wave amplitude and duration. Linear regressions between maximal M-wave variations and resting levels of TBARS and ORP were investigated.