Heart rate variability (HRV) is widely used to assess autonomic nervous system (ANS) function. It is traditionally collected from a dedicated laboratory electrocardiograph (ECG). This presents a barrier to collecting the large samples necessary to maintain the statistical power of between-subject psychophysiological comparisons. An alternative to ECG involves an optical pulse sensor or photoplethysmograph run from a smartphone or similar portable device: smartphone pulse rate variability (SPRV). Experiment 1 determined the simultaneous accuracy between ECG and SPRV systems in n = 10 participants at rest. Raw SPRV values showed a consistent positive bias, which was successfully attenuated with correction. Experiment 2 tested an additional n = 10 participants at rest, during attentional load, and during mild stress (exercise). Accuracy was maintained, but slightly attenuated during exercise. The best correction method maintained an accuracy of +/-2% for low-frequency spectral power, and +/-5% for high-frequency spectral power over all points. Thus, the SPRV system records a pulse-to-pulse approximation of an ECG-derived heart rate series that is sufficiently accurate to perform time- and frequency-domain analysis of its variability, as well as accurately reflecting change in autonomic output provided by typical psychophysiological stimuli. This represents a novel method by which an accurate approximation of HRV may be collected for large-sample or naturalistic cardiac psychophysiological research.