The study goal was to investigate autonomic activity with heart rate variability analysis during different sleep stages in males and females. The study utilized a 2 Groups (males, females) × 4 States (waking, stage 2 sleep, stage 4 sleep, rapid-eye movement sleep) mixed design with one repeated, within-subjects factor (i.e., state). The study was carried out in the sleep laboratory of the Thomas N. Lynn Institute for Healthcare Research. Twenty-four healthy adults (fourteen females and ten males). NA All participants underwent polysomnographic monitoring and electrocardiogram recordings during pre-sleep waking and one night of sleep. Fifteen-minute segments of beat-to-beat heart rate intervals during waking, stage 2 sleep, stage 4 sleep, and REM sleep were subjected to spectral analysis. Compared to NREM sleep, REM sleep was associated with decreased high frequency (HF) band power, and significantly increased low frequency (LF) to (HF) ratio. Compared to females, males showed significantly elevated LF/HF ratio during REM sleep. Males also demonstrated significantly decreased HF band power during waking when compared to females. No significant sleep- or gender-related changes in LF band power were found. The results confirmed changes in autonomic activity from waking to sleep, with marked differences between NREM and REM sleep. These changes were primarily due to stage-related alterations in vagal tone. REM sleep was characterized by increased sympathetic dominance, secondary to vagal withdrawal. The data also suggested gender differences in autonomic functioning during waking and sleep, with decreased vagal tone during waking and increased sympathetic dominance during REM sleep in the males.