OBJECTIVES: The effect of total sleep deprivation on heart rate variability (HRV) in groups of postmenopausal women on oral hormone therapy (HT) (on-HT, n = 10, 64.2 (1.4) years), postmenopausal women without HT (off-HT, n = 10, 64.6 (1.4) years) and young women (n = 11, 23.1 (0.5) years) was studied using a prospective case-control setup. METHODS: Polysomnography was performed over an adaptation night, a baseline night, and a recovery night after 40 h of total sleep deprivation. Time and frequency domain and nonlinear HRV from overnight electrocardiogram recordings were compared between groups during baseline and recovery nights. Further, the changes in HRV from baseline to recovery were analysed and compared between groups. Finally, correlations of HRV to percentages of sleep stages and measures of sleep fragmentation were analysed during baseline and recovery. RESULTS: Young women had higher HRV than older women; the most marked difference was between young and on-HT postmenopausal women. Sleep deprivation induced a decrease in frequency domain HRV in young and in off-HT women, an increase in alpha2 in off-HT women, and an increase in mean heart rate in on-HT women. The sleep deprivation effect was mainly uncorrelated to changes in sleep parameters. CONCLUSIONS: Acute total sleep deprivation has a deleterious effect on the autonomic nervous system in young women, but an even more pronounced effect in postmenopausal women. Hormone therapy use in late postmenopause does not give protection against these changes. These harmful effects may partly explain the increased cardiovascular morbidity and overall mortality associated with sleep loss.