Heart Rate Variability (HRV) analysis has become an important tool in assessing human Autonomic Nervous System (ANS) activity in recent years. Orthostatic challenge is one of the most common tests to detect ANS dysfunction. In this study we looked at the changes in ANS activity of normal subjects to orthostatic challenge and compared the results of 3 different HRV analysis methods: Time-Domain Methods, HRV spectral analysis without respiratory analysis (RA) and with RA. Although all three methods have indicated an increase in sympathetic activity and a decrease in parasympathetic activity from baseline to stand, the only significant increase in sympathetic activity was observed in HRV with RA method. Additional information from RA enables isolating the sympathetic and parasympathetic branches in HRV signals and therefore reflects ANS changes more accurately. On the other hand, sympathetic and parasympathetic power may not be separated properly if respiration-dependent fluctuations in HRV are ignored. It is expected that the differences between methods would be very clear with low respiratory rates. However, we focused on studies with normal respiratory rates and have also found significant differences among the methods.