Heart rate variability (HRV) is a particularly valuable quantitative marker of the flexibility and balance of the autonomic nervous system. Significant advances in software programs to automatically derive HRV have led to its extensive use in psychophysiological research. However, there is a lack of systematic comparisons across software programs used to derive HRV indices. Further, researchers report meager details on important signal processing decisions making synthesis across studies challenging. The aim of the present study was to evaluate the measurement fidelity of time-and frequency-domain HRV indices derived from three predominant signal processing software programs commonly used in clinical and research settings. Triplicate ECG recordings were derived from 20 participants using identical data acquisition hardware. Among the time-domain indices, there was strong to excellent correspondence (ICC avg =0.93) for SDNN, SDANN, SDNNi, rMSSD, and pNN50. The frequency-domain indices yielded excellent correspondence (ICC avg =0.91) for LF, HF, and LF/HF ratio, except for VLF which exhibited poor correspondence (ICC avg =0.19). Stringent user-decisions and technical specifications for nuanced HRV processing details are essential to ensure measurement fidelity across signal processing software programs.