Subtle alterations of the hypothalamic-pituitary-adrenal (HPA) axis in chronic fatigue syndrome (CFS) have been proposed as a shared pathway linking numerous etiological and perpetuating processes with symptoms and observed physiological abnormalities. Because the HPA axis is involved in the adaptive responses to stress and CFS patients experience a worsening of symptoms after physical and psychological stress, we tested HPA axis functioning with three centrally acting stress tests. We employed two procedures mimicking real life stressors and compared them with a standardized pharmacological neuroendocrine challenge test. A total of 21 CFS patients were compared with 25 healthy controls regarding their cardiovascular and endocrine reactivity in a psychosocial stress test and a standardized exercise test and their endocrine response in the insulin tolerance test (ITT). Controlling for possible confounding variables, we found significantly lower ACTH responses in the psychosocial stress test, the exercise test and in the ITT, with no differences in plasma total cortisol responses. In all tests, CFS patients had significantly reduced basal ACTH levels. Cardiovascular responses were not significantly different between the groups in the psychosocial stress test and the exercise test. These results suggest that CFS patients are capable of mounting a sufficient cortisol response under different types of stress, but that on a central level subtle dysregulations of the HPA axis exist. Interestingly, the observed extent of the endocrine alterations was significantly associated with the duration of the syndrome. A postulated deficient hypothalamic CRH secretion could serve as a neuroendocrine substrate of the observed fatigue and pain symptomatology in CFS. Furthermore, these endocrine alterations could be secondary to chronicity of the syndrome.