The aim of our study was to investigate the reciprocal interactions among sleep, ANS and occurrence of migraine attacks. We studied 8 consecutive patients with sleep-related migraine (two men and six women, mean age 41.9 ± 13.9), and high frequency of attacks (>5/month). Patients were evaluated during a headache-free period. Patient underwent full-night laboratory video-polysomnography and Heart Rate Variability (HRV) analysis and were compared with a large sample of normal subjects. For the HRV analysis, in time and frequency domain, we selected periods of 5 min period, from quiet wakefulness (W), stage 2 (N2) and 3 (N3) of N-REM, and REM sleep (R). We found a statistically significant reduction of LF/HF ratio during N2 and N3 sleep stages in migraineurs compared with controls. The ANS activity in migraineurs showed an higher level of fluctuation compared with normal subjects, with an increase of parasympathetic activation during N-REM sleep. This finding could be consequent to a subtle chronic sympathetic dysfunction which could manifest selectively, or become more evident, during sleep, and could represent a further measure of reduced arousability. The simultaneous involvement of the autonomic, arousal and pain systems suggest an involvement of the hypothalamic orexinergic pathways.