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Introduction: Multiple studies have shown that peak oxygen consumption is reduced in the majority of ME/CFS patients, using the golden standard for measuring exercise intolerance: cardiopulmonary exercise testing (CPET). A 2-day CPET protocol has shown different results on day 2 in ME/CFS patients compared to sedentary controls. No comparison is known between ME/CFS and idiopathic chronic fatigue (ICF) for 2-day CPET protocols. We compared ME/CFS patients with patients with chronic fatigue who did not fulfil the ME/CFS criteria in a male population and hypothesized a different pattern of response would be present during the 2nd day CPET. Methods: Fifty-one female patients with ICF completed a 2-day CPET protocol and were compared to an age/sex-matched group of 50 female ME/CFS patients. Measures of oxygen consumption (VO₂), heart rate (HR), systolic and diastolic blood pressure, workload (Work), and respiratory exchange ratio (RER) were collected at maximal (peak) and ventilatory threshold (VT) intensities. Results: Baseline characteristics for both groups were similar for age, BMI, BSA, and disease duration. A significance difference was present in the number of patients with fibromyalgia (seven ME/CFS patients vs zero ICF patients). Heart rate at rest and the RER did not differ significantly between CPET 1 and CPET 2. All other CPET parameters at the ventilatory threshold and maximum exercise differed significantly (p-value between 0.002 and <0.0001). ME/CFS patients showed a deterioration of performance on CPET2 as reflected by VO₂ and workload at peak exercise and ventilatory threshold, whereas ICF patients showed improved performance on CPET2 with no significant change in peak workload. Conclusion: This study confirms that female ME/CFS patients have a reduction in exercise capacity in response to a second day CPET. These results are similar to published results in female ME/CFS populations. Patients diagnosed with ICF show a different response on day 2, more similar to sedentary and healthy controls.