Sixteen ME/CFS patients were recruited via specialist clinics and ME/CFS support groups from the Adelaide, South Australia Greater Metropolitan area. Ten healthy participants were recruited to act as controls using convenience sampling from patient and research centre networks. Controls were matched to ME/CFS patients on the basis of age, body mass index (BMI) and physical activity status. All participants were required to be between the ages of 18–65 years, and ME/CFS patients had to have been previously diagnosed with ME/CFS based on one of three widely accepted diagnostic criteria: (1) 1994 Centers For Disease Control and Prevention (CDC 1994—also known as the ‘Fukuda’ criteria ), (2) 2003 ‘Canadian’ Consensus Criteria (CCC) , or (3) 2011 International Consensus Criteria (ICC) . With the exception of the patients being diagnosed with ME/CFS, all participants had to self-report as free of additional health conditions and injuries, and were required to be classed as low-moderate risk based on a self-report pre-exercise health screening  — all self-report screenings were conducted in the presence of an Accredited Exercise Physiologist, and any potential participants who were classified as moderate risk based on this screening process underwent additional screening to ensure that participation in the study represented minimal risk to the participant. All participants were required to be sedentary (< 150 min of moderate physical activity per week) and were excluded if they were taking any medication or had any known medical conditions (excluding ME/CFS) which could alter the response to exercise (e.g. beta-blockers, anti-depressants/postural orthostatic tachycardia syndrome). Experimental procedures were approved by the University of South Australia Human Research Ethics Committee and all participants provided written informed consent prior to participating.
Participants first attended the laboratory for an initial habituation session, during which they were familiarised with the laboratory and with the questionnaires and procedures to be used during the study. Participants first completed the Chalder Fatigue Scale, an 11 item questionnaire which measures the severity of fatigue, and has been previously validated in ME/CFS patients , before undergoing familiarisation with the exercise protocols to be used within the study. Given that ME/CFS patients typically experience a significant exacerbation of their symptoms following strenuous physical exertion, it was not feasible to expose participants to the full protocol used with the main testing sessions (which included a maximal exercise test) within the familiarisation session. Instead, participants were familiarised with the exercise equipment by performing 5 min of submaximal exercise on an electronically-braked cycle ergometer (Ergoselect 200, Ergoline GmbH, Bitz, Germany) while heart rate (HR) was measured using a HR monitor (RS800CX, Polar Electro Oy, Kempele, Finland), and were connected to an indirect calorimetry system (TrueOne 2400, Parvo Medics, East Sandy, Utah) via a two-way non-rebreathing value (Hans-Rudolph inc., Shawnee, Kansas). Throughout all testing sessions, efforts were made to minimise the amount of time that participants spent in waiting areas, and were given access to a reclining chair while waiting according to the recommendations of Stevens et al. .
2-day CPET protocol
Following the familiarisation session, participants returned to the laboratory at least 24 h later for the first of two exercise testing sessions, which consisted of a submaximal warm-up followed by CPET. Each session had an identical protocol and were performed on consecutive days. Participants first completed the Chalder Fatigue Scale before being fitted with the HR monitor and then resting supine for 10 min. Participants were then seated on the bicycle ergometer, fitted with the breathing valve for the indirect calorimetry system, and told to rest quietly while sitting on the bike. Following a seated rest period of 4–6 min, participants were instructed to commence cycling at a self-selected cadence for 5 min at 40 W for males and 30 W for females, which served as a warm-up. Following the initial 5 min of steady state exercise, the work rate was increased by 5 W increments every 20 s, until volitional exhaustion. All participants were given frequent verbal encouragement throughout the incremental portion of the test, to help elicit a maximal effort . Immediately following the cessation of exercise, participants were assisted to dismount the cycle ergometer and lay supine for 2 min. Ratings of perceived exertion (RPE) were collected at the end of each minute during the exercise test using Borg’s 6–20 RPE scale . Following the initial exercise test, participants returned to the laboratory at the same time on the following day, and the protocol was repeated in an identical fashion. Following each maximal exercise test, all participants were monitored whilst resting within the laboratory until they felt well enough to leave of their own accord. Additionally, participant well-being was monitored over the 2-weeks following the testing, to ensure no adverse events.
Peak oxygen uptake (peak V̇O2) was defined as the highest value for any 15-s epoch obtained during the exercise test. Determination of peak V̇O2 is typically done by identifying a plateau in V̇O2 in response to successive increments in workload, however this response is difficult to obtain in sedentary subjects . As a result, V̇O2 values were considered to have reached a valid maximal level if participants fulfilled two or more secondary criteria: (1) achievement of at least 90% of age predicted maximal HR , (2) respiratory exchange ratio (RER) > 1.1, and (3) RPE ≥ 17 . Participants’ data were excluded from the analysis if they were unable to produce a maximal effort based on these criteria. VT was calculated using the V-Slope method . Ventilatory data used for calculation of VT consisted of the final 30 s of steady state workload data, and all subsequent ventilatory data points.
All HR data were downloaded as R–R intervals to Polar Protrainer 5 software (Polar Electro Oy, Kempele, Finland), where artefacts or ectopic heartbeats were removed using the software’s automatic data filtering function. Resting HR (RHR) was defined as the average HR during the final 2 min of the pre-exercise supine rest. Steady state HR (SSHR) was defined as the average HR during the final 30 s of the 5-min steady state exercise which preceded the incremental portion of the testing. Peak HR was defined as the maximum HR produced during the maximal exercise test.
Statistical analysis was performed using IBM SPSS Statistics 21 (IBM Corporation, Armonk, NY). All data were checked for normality of distribution using the Shapiro–Wilk test prior to analysis. Unpaired t-tests were used to determine if there were any differences at baseline (Day 1) between patients and controls for any dependant variables. To determine the effect of post-exertional malaise on the dependant variables, two-way repeated measures ANOVA was performed to identify any main effects of group (patient or control), time (day 1 or day 2) or any group × time interaction effects. Where significant main effects were identified, estimated marginal means were assessed to determine where those differences occurred. To determine if any of the assessed parameters represent a useful tool to aid in differentiating between ME/CFS patients and controls, receiver operator characteristic (ROC) analysis was conducted on any objective physiological variables which demonstrated a significant group or group × time interaction in order to compute sensitivity and specificity of these variables for differentiating ME/CFS participants from controls. Statistical significance was set at p < 0.05. All data are presented as mean ± standard deviation (SD).