Introduction: Pulse rate variability (PRV) refers to the changes in pulse rate through time and is extracted from pulsatile signals such as the photoplethysmogram (PPG). Although PRV has been used as a surrogate of heart rate variability (HRV), which is measured from the electrocardiogram (ECG), these variables have been shown to have differences, and it has been hypothesised that these differences may arise from technical aspects that may affect the reliable extraction of PRV from PPG signals. Moreover, there are no guidelines for the extraction of PRV information from pulsatile signals. Aim: In this study, the extraction of frequency-domain information from PRV was studied, in order to establish the best performing combination of parameters and algorithms to obtain the spectral representation of PRV. Methods: PPG signals with varying and known PRV content were simulated, and PRV information was extracted from these signals. Several spectral analysis techniques with different parameters were applied, and absolute, relative and centroid-related frequency-domain indices extracted from each combination. Indices from extracted and known PRV were compared using factorial analyses and Kruskal-Wallis tests to determine which spectral analysis technique gave the best performing results. Results: It was found that using fast Fourier transform and the multiple signal classification (PMUSIC) algorithms gave the best results, combined with cubic spline interpolation and a frequency resolution of 0.0078Hz for the former; and a linear interpolation with a frequency resolution as low as 1.22 × 10-4, as well as applying a fifth order model, for the latter. Discussion: Considering the lower complexity of FFT over PMUSIC, FFT should be considered as the appropriate technique to extract frequency-domain information from PRV signals.