Hypothalamus-pituitary-adrenal (HPA) axis activity was monitored in 20 women with chronic migraine (CM), previously affected by medication overuse headache (MOH), in comparison to healthy women (20 subjects) by measuring salivary cortisol, testosterone, dehydroepiandrosterone-sulphate (DHEA-S) levels, and their ratios, one week after the end of the MOH rehabilitation procedure. The participants were instructed how to collect saliva samples at home, a procedure that was performed twice a day (08:00 a.m. and 8:00 p.m.). Morning and evening levels of cortisol were significantly increased in CM patients with respect to controls. With regard to the cortisol/DHEA-S ratio, an inverse marker of psycho-physical wellbeing, CM women showed significantly higher values than controls. Moreover, testosterone/cortisol ratios (anabolic/catabolic index of physical performance) were significantly lower in CM patients than in controls. In the present study, CM appears not to be associated with an impairment of cortisol and DHEAS circadian fluctuation; however, CM patients present alterations in HPA axis function that might contribute to metabolic and psychological alterations that have also been associated with CM.