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The physiology of menopausal hot flashes is not well understood. The autonomic nervous system may play a role in hot flashes, but the current understanding is limited. We previously demonstrated in the laboratory that decreases in high-frequency heart rate variability, an index of cardiac vagal control, occur during hot flashes relative to preceding and following periods. In the present study, we tested whether we would observe a similar phenomenon in the ambulatory setting. We additionally considered respiratory rate in these associations.Twenty-one perimenopausal and postmenopausal women aged 40 to 60 years reporting daily hot flashes were monitored both for physiologic and reported hot flashes and heart rate variability during a 24-hour period as they went about their daily lives. Heart rate variability estimates were derived using the band-limited variance method. The interval during the hot flash was compared with two nonflash periods before and after the hot flash via mixed-effects models.Heart rate variability significantly decreased during hot flashes relative to periods preceding (B = 0.31, SE = 0.03; P < 0.0001) and following (B = 0.30, SE = 0.03; P < 0.0001) physiologic hot flashes (covariates: age, race, education, menopause status, physical activity, body mass index, and anxiety). The findings were comparable considering self-reported hot flashes. They additionally persisted when controlling for respiratory rate.Significant decreases in cardiac vagal control occurred during hot flashes assessed during women’s daily lives. These findings extend our work in the laboratory to the ambulatory setting, further shedding light on the physiology of hot flashes and underscoring a potential role of parasympathetic function in hot flashes.