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Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) have been shown to exhibit altered ventilatory characteristics on the second of two progressive maximal cardiopulmonary exercise tests (CPET) performed on consecutive days. However, maximal exercise can exacerbate symptoms for ME/CFS patients and cause significant post-exertional malaise. Assessment of heart rate (HR) parameters known to track post-exertional fatigue may represent more effective physiological markers of the condition and could potentially negate the need for maximal exercise testing. Sixteen ME/CFS patients and 10 healthy controls underwent a sub-maximal warm-up followed by CPET on two consecutive days. Ventilation, ratings of perceived exertion, work rate (WR) and HR parameters were assessed throughout on both days. During sub-maximal warm-up, a time effect was identified for the ratio of low frequency to high frequency power of HR variability (p=0.02) during sub-maximal warm-up, and for HR at ventilatory threshold (p=0.03), with both being higher on Day Two of testing. A significant group (p