Heart rate variability (HRV) is commonly used within sleep and cardiovascular research, yet HRV reliability across various sleep stages remains equivocal. The present study examined the reliability of frequency- and time-domain HRV within stage-2 (N2), slow-wave (SWS), and rapid-eye-movement (REM) sleep during both stable and disrupted sleep. We hypothesized that high-frequency (HF) HRV would be reliable in all three sleep stages, low-frequency (LF) HRV would be reliable during N2 and SWS, and that disrupted sleep via spontaneous cortical arousals would decrease HRV reliability. Twenty-seven participants (11 men, 16 women, 26 +/- 1 yr) were equipped with laboratory polysomnography for 1 night. Both frequency- and time-domain HRV were analyzed in two 5- to 10-min blocks during multiple stable and disrupted sleep cycles across N2, SWS, and REM sleep. HF HRV was highly correlated across stable N2 (r = 0.839, P < 0.001), SWS (r = 0.765, P < 0.001), and REM (r = 0.881, P < 0.001). LF HRV was moderate-to-highly correlated during stable cycles of N2 sleep (r = 0.694, P < 0.001), SWS, (r = 0.765, P < 0.001), and REM (r = 0.699, P < 0.001) sleep. When stable sleep was compared with disrupted sleep, both time- and frequency-domain HRV were reliable (alpha > 0.90, P < 0.05) in N2, SWS, and REM, except for LF HRV during SWS (alpha = 0.62, P = 0.089). In conclusion, time- and frequency-domain HRV demonstrated reliability across stable N2, SWS, and REM sleep, and remained reliable during disrupted sleep. These findings support the use of HRV during sleep as a tool for assessing cardiovascular health and risk stratification.NEW & NOTEWORTHY Heart rate variability (HRV) is a commonly employed indirect estimate of cardiac autonomic activity during sleep with limited reliability studies. Nocturnal frequency-domain HRV was reliable across differing stable sleep cycles of stage-2 (N2), slow-wave (SWS), and rapid-eye-movement (REM) sleep. Moreover, frequency- and time-domain HRV were reliable during stable and disturbed sleep, except SWS low-frequency HRV. Our finding supports nocturnal HRV as a potential tool for cardiovascular risk stratification.