Background <p>Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating but poorly-understood disease. ME/CFS symptoms include immune system effects alongside incapacitating fatigue and post-exertional disease exacerbation. Symptom severity can range from mild to severe and whilst symptoms can fluctuate, few people fully recover.</p> Methods <p>Immunological profiles of people living with ME/CFS were analysed by flow cytometry, focusing on cytotoxic cells, to determine whether people with mild/moderate (<i>n</i> = 43) or severe ME/CFS (<i>n</i> = 53) expressed different immunological markers. Flow cytometry data were tested for normality and the two clinical groups were compared by t-test or Mann-Whitney U-test as appropriate.</p> Results <p>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, indicative of persistent viral infection. 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 interferon-γ, tumour necrosis factor and interleukin-17, following stimulation in vitro, indicative of prolonged non-specific inflammation. 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.</p> Conclusions <p>These immunological differences likely reflect different disease pathogenesis mechanisms occurring in the two clinical groups, opening up opportunities for the development of prognostic markers and stratified treatments.</p>