OBJECTIVE This study was conducted to test the hypothesis of autonomic imbalance in overtrained athletes during sleep and after awakening with analyses of heart rate variability (HRV) and nocturnal urine stress hormones. METHODS We examined 12 athletes diagnosed to be severely overtrained (OA, 6 men and 6 women, mean age (+/-SD) 25 +/- 7 yr) and 12 control athletes (CA, 6 men and 6 women, mean age 24 +/- 5 yr). Overtraining diagnosis was further supported by higher perceived stress in OA than in CA (24.8 +/- 10.8 vs 15.3 +/- 5.5, P < 0.05). HRV was analyzed with time and frequency domain methods from RR intervals (RRI) recorded during sleep and after awakening. Nocturnal urine stress hormones were analyzed by liquid chromatography. RESULTS No differences were found in HRV or stress hormones during night sleep. After awakening, the standard deviation of RRI (84 +/- 31 vs 116 +/- 41 ms, P < 0.05) and low-frequency power of RRI (2153 +/- 2232 vs 4286 +/- 2904 ms, P < 0.05) were lower in OA than in CA. From sleep to after awakening, the coefficient of variation of RRI decreased more in OA than in CA (from 11.8 +/- 3.3 to 7.7 +/- 2.5%, P < 0.001 vs from 11.9 +/- 1.8 to 10.0 +/- 2.5%, P < 0.01, respectively, interaction P < 0.05). CONCLUSIONS The present findings suggest that in OA, cardiac autonomic modulation is at the level of control athletes during sleep, but the parasympathetic cardiac modulation is slightly diminished after awakening. Further investigations should concentrate on autonomic responses to different challenges, such as awakening in the present study.