Sleep deprivation (SD) leads to cardiovascular risk by disturbing autonomic nervous system (ANS). Whether sex or menstrual cycle influences cardiac ANS interfered by SD remained unclear. This investigation was to further clarify the effects of different menstrual phases on cardiac autonomic nervous activity disturbed by SD. Methodologically, cardiac autonomic modulation before and after 30 h of total SD was determined by spectral analysis of heart rate variability in 10 healthy females during the mid-follicular (FF) and mid-luteal (FL) phases and 10 healthy males. The result showed that before SD, the FF subjects, but not FL, had higher normalized high frequency (nHF) (vagal activity) or lower ratio of low frequency (LF) to high frequency (HF) power (sympathetic activity) while performing deep breathing or Valsalva maneuver (40 mmHg) than the male subjects did. However, the SD for 30 h in the FF group significantly increased the LF/HF ratio and decreased nHF level at rest and in responses to deep breathing and Valsalva maneuver, despite no significant change of HRV in either male or FL group. Simultaneously, the SD substantially lowered venous PCO_2 in the FF group, and the decreased PCO_2 level was associated with the increased LF/HF ratio following SD. We concluded that 30-h acute SD promotes sympathetic and depress parasympathetic activities in female during the follicular phase rather than the luteal phase. Compared to FF, SD-triggered cardiac sympathetic activation is blunted in FL. The study provides further insight into the physiology of acute SD in different sex and menstrual phases.