BACKGROUND: The mechanisms of acetazolamide (ACZ) in the prophylaxis of acute mountain sickness (AMS) remain unclear. This study evaluated the changes in physiological variables of sleep and heart rate variability (HRV) in subjects with earlier history of AMS who underwent prophylactic treatment of ACZ. METHODS: Nonacclimatized healthy subjects were transported using a bus from 555 m to 3150 m within 3 hours. Polysomnography (PSG) was performed 3 days before ascent (T0), for two consecutive nights at 3150 m (T1 and T2), and 2 days after descent (T3). HRV was measured before sleep and after awakening from T0 to T3. AMS was diagnosed using a self-reported Lake Louise score questionnaire. Subjects found confirmed to have AMS were enrolled in this study. The physiological variables and HRV were compared in AMS subjects without (control group) and with prophylactic ACZ (prophylactic ACZ group). RESULTS: Thirteen AMS subjects were enrolled. The PSG results were analyzed in eight and HRV were analyzed in nine of the 13 subjects. The prophylactic use of ACZ in the subjects with a history of AMS significantly improved sleep efficiency (p = 0.012) and awakening percentages (p = 0.017) at T1, significantly higher levels of arterial oxygen saturation (SaO2) and lower values of partial pressure end-tidal carbon dioxide tension (PETCO2) at four time points. Furthermore, they had a higher rapid eye movement sleep percentage (p = 0.05) at T2. Prophylactic ACZ treatment significantly increased the normalized unit of high frequency at T1 after awakening (p = 0.028). CONCLUSION: Significantly higher quality of sleep, higher SaO2 during sleep, and lower PETCO2 at high altitude were found in the subjects with a history of AMS using prophylactic ACZ before rapid ascent. ACZ may accelerate the acclimatization process for rapid ascents to high altitudes by increasing parasympathetic tone based on HRV analyses.