Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating but poorly-understood disease. ME/CFS symptoms can range from mild to severe, and include immune system effects alongside incapacitating fatigue and post-exertional disease exacerbation. In this study, we examined immunological profiles of people living with ME/CFS by flow cytometry, focusing on cytotoxic cells, to determine whether people with mild/moderate (n= 43) or severe ME/CFS (n=53) expressed different immunological markers. We found that people with mild/moderate ME/CFS had increased expression of cytotoxic effector molecules alongside enhanced proportions of early-immunosenescence cells, determined by the CD28-CD57- phenotype. In contrast, people with severe ME/CFS had higher proportions of activated circulating lymphocytes, determined by CD69+ and CD38+ expression, and expressed more pro-inflammatory cytokines, including IFNγ, TNF and IL-17, following stimulation in vitro. These changes were consistent across different cell types including CD8+ T cells, mucosal associated invariant T cells and Natural Killer cells, indicating generalised altered cytotoxic responses across the innate and adaptive immune system . These immunological differences likely reflect different disease pathogenesis mechanisms occurring in the two clinical groups, and opening up opportunities for the development of prognostic markers and stratified treatments. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The analysis was mainly funded by the NIH: R01AI103629, with additional support from NIH: 5R01AI170839-03 and from The ME Association. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted according to the principles of the Declaration of Helsinki. Study approval was obtained from the LSHTM Medicine Research Ethics Committee 16 January 2012 (Ref.6123) and the National Research Ethics Service (NRES) London: Bloomsbury Research Ethics Committee 22 December 2011 (REC ref.11/10/1760, IRAS ID: 77765). Written informed consent was obtained from all study participants prior to inclusion in the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data underlying the analysis can be accessed in the Supplementary File contained within the manuscript.