BACKGROUND: Heart rate variability (HRV) is a popular tool to quantify autonomic function. However, this typically requires an expensive 3-12 lead electrocardiogram (ECG) and BioAmp system. This investigation sought to determine the validity and reliability of an OpenBCI cyton biosensing board (open source) for accurately quantifying HRV. NEW METHOD: A cyton board with a 3-lead ECG was employed to acquire heart rate waveform data, which was processed to obtain HRV within both time- and frequency-domains. The concurrent validity was compared to a simultaneous recording from an industry-standard 3-lead ECG (ADInstruments) (n = 15). The reliability of the cyton board was compared between three days within a 7-day timespan (n = 10). Upright quiet-stance short-term HRV metrics were quantified in time- and frequency-domains. RESULTS: The two devices displayed excellent limits of agreements (all log mean differences +/-0.4) and very high between-device variable associations (all r (2) > 0.98). Between the three time points in the same subjects, no differences were noted within time- (all p > 0.71) or frequency-domains (all p > 0.88) across testing points. Finally, all HRV metrics exhibited excellent levels of reliability through high Cronbach\'s Alpha (all >/=0.916) and intraclass correlation coefficients (all >/=0.930); and small standard error of the measurement (all </=0.7) and typical error of the measurement (all </=0.1) metrics. COMPARISON WITH EXISTING METHODS: The cyton board with 3-lead ECG was compared with an industry-standard ADInstruments ECG during HRV assessments. There were no significant differences between devices with respect to time- and frequency-domains. The cyton board displayed high-levels of between-day reliability and provided values harmonious to previous ECG literature highlighting the applicability for longitudinal studies. CONCLUSION: With proper background knowledge regarding ECG principles and a small degree of set-up complexity, an open source cyton board can be created and employed to perform multimodal HRV assessments at a fraction of the cost (~4%) of an industry-standard ECG setup.