Although patterns of heart rate variability (HRV) hold considerable promise for clarifying issues in clinical applications, the inappropriate quantification and interpretation of these patterns may obscure critical issues or relationships and may impede rather than foster the development of clinical applications. The duration of the RR interval series is not a matter of convenience but a fine balance between two important issues: acceptable variance and stationarity of the time series on one hand, and acceptable resolution of the spectral estimate and reduced spectral leakage on the other. Further, in the standard short-term HRV analysis, it has been observed that the previous studies in HRV spectral analysis use a wide range of RR interval segment duration for spectral estimation by Welch\'s algorithm. The standardization of RR interval segment duration is also important for comparisons among studies and is essential for within-study experimental contrasts. In the present study, a comparative analysis for RR interval segment durations has been made to propose an optimal RR interval segment duration. Firstly a simulated signal was analyzed with Hann window and zero padding for the segment lengths of 1024, 512, 256 and 128 samples resampled at 4 Hz with 50% overlapping. Again, the above procedure was applied to RR interval series and it was concluded that segment length of 256 samples with 50% overlapping provides a smoothed spectral estimate with clearly outlined peaks in low- and high-frequency bands. This easily understandable and interpretable spectral estimate leads to a better visual and automated analysis, which is not only desirable in basic physiology studies, but also a prerequisite for a widespread utilization of frequency domain techniques in clinical studies, where simplicity and effectiveness of information are of primary importance.