Autonomic nervous system (ANS) acts as a control system functioning largely below the level of consciousness, and controls visceral functions. The activity of the ANS has been assessed by means of the heart rate variability (HRV). It has been reported that HRV is dependent on sex, age, body mass index, and smoking, etc. However, the effects of posture and sleep deprivation on HRV have rarely been reported. Objective of our work was to find out which posture is appropriate for stable HRV. We measured the number of sleep deprivation and HRV using power spectrum in six stages for 30 minutes. Increased low frequency (LF) power and high frequency (HF) power indicate enhanced sympathetic and parasympathetic activity, respectively. We determined the LF/HF ratio to minimize individual difference. It was found that sleep deprivation by awakening up subjects was affected by posture, which resulted in changes of LF/HF. Although LF/HF varied with time, it was more stable in sitting than in supine. In conclusion, we recommend sitting posture when measuring HRV because of less sleep deprivation resulting in less variation in LF/HF.