ME/CFS is a major public health problem significantly impairing quality of life. Although efforts have been made to refine the diagnostic criteria and definition of ME/CFS, studying this syndrome remains challenging because of the heterogeneity in presentation, variability in ME/CFS duration and severity and absence of a reliable diagnostic laboratory test or biomarker10,11. In this study, we focused on a carefully selected group of ME/CFS patients with significant post-exertional malaise but still able to exercise. Our study has three main findings. First, we have found that exercise can be associated with significant changes in cytokine profile that are still observed 18 hours following symptom-limited exercise. Second, our study suggests that exercise may allow better discrimination of ME/CFS case status than resting values. Third, we have found that cardiac structure at baseline and cardiorespiratory responses following exercise with a one-day protocol do not appear to distinguish cases of ME/CFS from healthy sedentary controls.

Previous studies have analyzed changes in selected cytokines and growth factors profiles but mainly focused on changes following strenuous exercise in athletic participants. Pedersen et al. and Toft et al. have shown that after a marathon TNF-α and IL-1β levels increase twofold and IL-6 levels increase up to 100-fold but decreases rapidly; this is followed by a marked increase in the concentration of IL-1RA and other anti-inflammatory or regulatory proteins such as IL-8 and IL-1012,13,14. In a recent small study on 10 sedentary individuals, Landers-Ramos et al. found that acute exercise (30 minutes of treadmill running at 75% of the subject’s peak VO 2 ) increased circulating concentrations of the angiogenic cytokines placental growth factor (PlGF), basic fibroblast growth factor (bFGF) and soluble fms-like tyrosine kinase-1 (sFlt-1), as well as IL-6 and IL-8 in sedentary young men15. In addition to the previous angiogenic growth factors, changes in VEGF have not been as extensively studied in healthy participants.

In our study we observed changes in cytokine profiling 18 hours post exercise in both healthy controls and patient with ME/CFS. The biological variability demonstrated in our study has significant implications for the field of cytokine profiling. Greater attention to recent bouts of exercise or activity level should be given as these variables may have implication for data interpretation.

An emerging hypothesis regarding the cause of ME/CFS is immune dysregulation, thought to be reflected in up-regulated pro-inflammatory cytokines leading to the symptoms that are characteristic of this illness. As highlighted by Hornig et al. in a large multicenter study (n = 298), cytokine expression in ME/CFS may vary according to the duration of symptoms, stratified in the study to 3 years; expression of cytokines could also vary depending on severity of symptoms. In their study CD40L (a protein of the TNF-receptor superfamily) and platelet-derived growth factor (a growth factor the regulates cell growth and division) were reduced in short duration disease subjects when compared to controls8. In contrast Nakamura et al. did not observe dynamic change in cytokine profile after exercise or sleep deprivation in a cohort of 26 females with ME/CFS. However, the levels of IL-1β, a cytokine that is part of the inflammasome complex which is often activated in response to metabolic or infectious stress, were higher at both baseline and during exercise in patients with ME/CFS16. A further case control study including 24 patients with ME/CFS undertaken by Clark et al. was unable to identify meaningful changes in select cytokines post a bout of exercise17.

Our study builds on these previous findings adding in terms of originality through the use of a comprehensive immune and growth factor panel (51-plex), a larger cohort of sedentary individuals (in comparison to previous studies) and analysis of persistent changes in circulating factors at 18 hours post exercise. By matching ME/CFS cases and healthy controls samples from day 1 and 2 on the same plate, we minimized the effect of inter-plate variability, in turn providing greater possibility of detecting significant dynamic changes within both cohorts. We found that acute exercise influenced several pathways including inflammatory, growth factors, stem cell factors and vascular factors, some of which persisted up to 18 hours. Consistent with previous exercise studies, elevation of TNF-α post exercise was seen in our sedentary controls, however, no change in IL-6, likely explained by its rapid decrease post exercise and potentially the lower signal to noise ratio of IL-6 on the 51-plex assay. There was also an increase in selected pro-inflammatory cytokines such as IL-2, IL-12p40 and TNF-α in our control group. We applied a network estimation algorithm which is useful for retaining connections between cytokines that are biological significant and removing connections that are statistical noise. Using this method, we found factors with exercise were richly connected, particularly IL-4, which is known to play a key role in immune regulation, specifically Th2 cells. The direct relationship between cytokines however, differed between case and controls networks supportive of a distinct cytokine inflammatory signature in ME/CFS.

Compared to resting cytokine profiles, our study highlights that post-exercise profiling could have greater value in discriminating case status than resting parameters. Among cytokines and growth or vascular factors identified in our discriminatory analyses, CD40L appeared to strongly contribute to discrimination, with negative correlation both 18 hours post exercise and through its change from baseline. This is consistent with previous findings indicating that a failure to reduced levels of CD40L post exercise is associated with increased symptom flare post a bout of moderate exercise18. The association with CD40L was also found in a recent larger study, at rest by our group (with no overlap of patients) with a trend for lower levels of CD40L compared to controls across the spectrum of disease severity19.

CXCL1, like CD40L contributed strongly to multivariable discrimination of cases and controls post exercise. Unlikely CD40L, in univariate analysis separately by cytokine, CXCL1 decreased with exercise in controls and cases. CXCL1 also demonstrated high relative centrality within the network participants with ME/CFS. Interestingly, increased CXCL1 production by neutrophils has been seen in patients with fibromyalgia, however, considering the small numbers within the studies, these findings should be considered exploratory and further investigation is required to define their clinical implications20.

Further supportive of an immune mediate pathway in ME/CFS, we found CXCL10 played a central role in the cytokine network and contributed to case discrimination when combine with delta change in IL-4, G-CSF, IL-1β, IL-7 and CD40L. Recently CXCL10 has been shown to play a role in autoimmune disease, in particular type 1 diabetes and inflammatory bowel disease, through the augmentation of the Th1 autoimmune response21,22. Further studies will be required to define its contribution to ME/CFS.

Several small studies initially suggested differences in cardiac size between ME/CFS and healthy controls3,4. Using patients well matched for level of activity, we were unable to find significant differences in cardiac structure or function in our ME/CFS cohort with regard to fitness independent measures of ventricular remodeling such as mass to volume ratio or scaled ventricular dimension. Similarly, we were unable to identify differences in vascular stiffness using central aortic pulse wave velocity or significant differences in endothelial function, peak VO 2 or ventilatory efficiency. The fact that no difference was detected between CPET parameters between both groups despite marked difference in MFI-20 scores highlights the difference between reported symptoms such as fatigue or dyspnea and exercise performance measured by peak VO 2 23. This underscores the importance of not using these two concepts interchangeably.

Regarding exercise protocol our study used a single submaximal exercise protocol with repeat blood draw 18 hours post exercise to correlate with the onset of post exercise malaise, optimize processing of serum samples and to determine whether cytokine profiling could better discriminate than CPET parameters on day 1. However, in 2007 a seminal study by Snell et al. demonstrated the value in using a two day CPET protocol, through diminished CPET performed a day after the first24. Contemporary studies have confirmed these findings and suggested the use of a two-day CPET challenge protocol when assessing patients with ME/CFS in particularly those with post exercise malaise25,26,27.

Our study has several limitations. First, although our patients were carefully selected and matched with sedentary controls, the sample size is small with a small but statistically significant difference in BMI between groups. There was however, no difference in the number of participants, overweight or obese by this classification. In an effort to avoid false discovery rates, we also conducted careful adjustment of multiple measures. The fact that exercise was able to reveal similar factors of the larger resting study of Hornig et al. increased confidence in our findings. Despite no exercise validation cohort, the fact that several factors discussed emerged in both patients with ME/CFS and sedentary controls also brings more confidence in the results. Luminex assays are also known not to have a good signal to noise ratio for IL-6, however, our findings appear consistent with contemporary studies. Finally, we selected a sub-group of patients with ME/CFS, with severe post exercise fatigue to ensure a more specific phenotype.

In conclusion, our study suggests that exercise may be useful to profile key biological difference in ME/CFS and sedentary controls. We also highlight the importance to account for exercise when profiling disease states or syndromes. Replicating the findings and investigating profiling using a two-day protocol will be important steps for future research.