This study aimed to (1) determine if 2-day CPET is a useful method for differentiating between ME/CFS patients and healthy controls, and (2) establish cut-off values which can be used to aid in the diagnosis of ME/CFS. The main finding of the present study was that during the 2nd day of the 2-day CPET, WR at VT was decreased for ME/CFS patients, confirming previous findings that an earlier onset of VT during the presence of post-exertional malaise is a potential biomarker of ME/CFS [8, 10, 11]. The change in WR at VT was paired with larger self-reported increases in fatigue in ME/CFS patients compared with controls on Day 2, while there were no differences in peak WR or peak VO 2 from Day 1 to Day 2 for either patients or controls. ROC analysis identified that the decrease in WR at VT of 6.3–9.8% on the 2nd day of 2-day CPET may represent an objective biomarker of ME/CFS, with high sensitivity and specificity.

In the current study, there were no differences between patients and controls for RHR, SSHR, peak HR, peak V̇O 2 or peak WR, suggesting that the patients and controls were well matched for their general fitness levels. Previous literature has suggested that ME/CFS patients may be affected by significant deconditioning compared to healthy, sedentary individuals [24], or kinesiophobia [25, 26], which may impact on their ability to produce a maximal effort. However, given the similarities between the patients and controls for peak V̇O 2 , SSHR, peak HR, and that all ME/CFS patients were able to produce a criteria-defined valid maximal effort, this does not appear to be the case in the current study. These findings are in agreement with the those of Sargent et al. [27] who reported no difference in V̇O 2 max between ME/CFS patients and controls when accepted criteria were applied to define the attainment of a maximal value. Importantly, the fact that ME/CFS patients produced the same WR and V̇O2 as controls on both days of testing implies that the change in any ventilatory or HR parameters as a result of the 2-day CPET were not due to a change in physical performance in the ME/CFS patients.

Receiver operator characteristic analysis performed on the change in WR at VT between Day 1 and Day 2 of testing revealed that an absolute decrease in WR at VT of 7.5–12.5 W or a percentage decrease of between − 6.3% and − 9.8% provided optimal specificity and sensitivity for differentiating between patients and controls. However, given the large variation in WR at VT for ME/CFS patients (ranging from 50 to 140 W in the current study), it is likely more appropriate to use the percentage, rather than absolute change in VT, as a method for differentiating between the two groups. It is important to acknowledge that CPET may exacerbate symptoms of post-exertional malaise and is therefore not an ideal method for determining the presence of ME/CFS, and markers that can be assessed under resting conditions would be preferable. However, while not ideal, evaluation of the change in WR at VT during 2-day CPET may be a valid and sensitive marker that can be used to aid in the diagnosis of ME/CFS. Based on the ROC analysis performed in the present study, we would propose that the more conservative measure of at least a 9.8% reduction in WR at VT be used for diagnosis, given this provided 100% specificity, indicating that a reduction of this magnitude is not likely to occur in a person who does not have ME/CFS.

Numerous previous studies have also found an earlier onset of VT in ME/CFS patients on the 2nd day of 2-day CPET. VanNess, Snell and Stevens [10] found a 30% reduction in V̇O2 at VT from the first to the 2nd day, while Keller, Pryor and Giloteaux [11] found a 15% decrease in V̇O 2 at VT and a 21% decrease in WR at VT, Snell et al. [8] found a 55% decrease in WR at VT and Hodges, Nielsen and Backen [14] found a 12% decrease in WR at VT for ME/CFS patients while controls experienced a 9% increase. While these results suggest that ME/CFS patients experience an earlier occurrence of VT on the 2nd day of 2-day CPET, the mechanism responsible for this effect is currently unknown. Potentially, the alterations in VT during the 2-day CPET may result from metabolic abnormalities. Metabolic abnormalities have been reported in skeletal muscle of ME/CFS patients related to impairment of oxygen delivery to skeletal muscle during exercise and inability to recover from exercise-induced reductions in pH [28,29,30]. Derangement of pH homeostasis might have resulted in a more rapid decrease in blood pH during the 2nd day of exercise in the present study. This would in turn lead to an increased CO 2 production through buffering of H+ ions by carbonic anhydrase, leading to stimulation of chemoreceptors with a subsequent increase in ventilation. However, while some studies have reported derangement of pH homeostasis in patients with ME/CFS, others have found such abnormalities are present in less than 50% of patients [31, 32]. In the present study though, VT occurred earlier in all but one ME/CFS patients (i.e. in 15/16 patients). Keller et al. [11] found a decrease in maximal O 2 pulse during the 2nd day of their 2-day CPET, indicating a compromised oxygen delivery in ME/CFS patients in the presence of post-exertional malaise, while Vermeulen et al. [12] found a non-significant 5% decrease in the same parameter on the 2nd day of testing. A lack of O 2 delivery/uptake may lead to an earlier transition to anaerobic energy systems and therefore a more rapid increase in lactic acid production and reduction in pH. Future studies employing a 2-day maximal testing protocol in patients with ME/CFS should endeavour to measure lactic acid and pH. Interestingly, the change in WR at VT from Day 1 to Day 2 in the current study was smaller than that seen in previous studies which have assessed this parameter in ME/CFS patients [8, 10, 11]. This is likely due to a difference in the protocols used to elicit a maximal effort. In the current study all participants were given regular verbal encouragement to help elicit a valid maximal effort. Conversely, it was not explicitly stated if verbal encouragement was provided in two of the three pervious studies [10, 11], and it has been shown that frequent verbal encouragement results in higher peak WR’s and prolonged maximal exercise tolerance [18] than when no encouragement is provided. This potential lack of encouragement may have resulted in a premature cessation of exercise on Day 2 of the 2-day CPET in ME/CFS patients, resulting in a greater exacerbating in the change in WR at VT.

This study is limited by a possible selection bias as a result of the 2-day CPET protocol used within the study. Given the potential for symptom exacerbation as a result of the 2-day CPET, patients with severe ME/CFS may be less likely to volunteer, whereas patients with mild-moderate ME/CFS may be more likely. Anecdotally, none of the included participants classified themselves as a severe sufferer of the condition, and so the inclusion of patients with a more severe form of ME/CFS may have produced a different result. Future research should attempt to include sufferers with severe ME/CFS in the study design; however the authors acknowledge the potential for severe symptom exacerbation for these people and hence the clinical and ethical considerations. Additionally, ME/CFS patients exhibited a wide range of time periods since initial diagnosis (ranging from 2 to 18 years). This may have affected the results as patients who have been suffering from the condition for longer periods may experience increased levels of physical detraining that are common in ME/CFS patients compared to those who had been diagnosed more recently.