BACKGROUND: Exercise activates hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system (ANS) and generally causes beneficial changes in homeostatic balance. However, the health benefits of late-night exercise programs on the activity of HPA and ANS is not known. The aim of this study was to assess effects of late-night exercise on sleep quality and on the activities of the HPA axis (as cortisol awakening response, CAR) and the ANS (as heart rate variability, HRV) measurements in the following morning. METHODS: Medical students (N.=20 males, 20-24 years old) filled Karolinska Sleep Diary on the day before exercise program. In the following morning, they provided salivary samples for the assessment of CAR (samples at 0, 15, 30 and 60 min postawakening) and had a 5-min electrocardiogram recording for the determination of HRV. In the next night, an exercise program consisting of a 90-min football match was implemented at 09:30 p.m. and all procedures were repeated. Cortisol concentrations were measured in the salivary samples and time- and frequency-domain parameters of HRV were calculated. RESULTS: Late-night exercise did not affect (P>0.05) sleep parameters (sleep duration, disturbed sleep, awakening problems) and CAR parameters (0, 15, 30, 60 min cortisol concentrations, mean concentration, area under the curve) but influenced HRV parameters (P<0.05). CONCLUSIONS: The results suggest that late-night exercise is associated with changed HRV activity rather than changes in CAR and, therefore, it might be suggested that late-night exercise affects ANS activity rather than HPA activity in the next morning.