Heart rate variability (HRV) is a psychophysiological phenomenon with broad implications, providing an accessible index of vagal function, underpinning psychological constructs, including the capacity for social engagement and emotion regulation, and may predict future morbidity and mortality. However, the lack of reference values for short-term HRV indices for participants of both sexes across the age spectrum is a limiting factor. This was the objective of the present study. Resting electrocardiographic records were obtained from 13,214 participants (both sexes, 35-74 years), and HRV indices in time and frequency domains (mean +/- SD) were determined from 5-min records. Results were based on a subsample of 2,874 nonmedicated, healthy participants stratified by sex across 10-year age groupings. Men showed lower heart rate (HR, 64 +/- 8 bpm vs. 68 +/- 8 bpm, p < .05) and normalized high frequency (HF; 39.4 +/- 18.0 normalized units [n.u.] vs. 50.4 +/- 18.5 n.u., p < .05) than women, and higher N-N variance (2,214 +/- 1,890 ms(2) vs. 1,883 +/- 1,635 ms(2) , p < .05), standard deviation of all N-N intervals (SDNN; 43.7 +/- 17.3 ms vs. 40.3 +/- 15.8 ms, p < .05) and LF/HF (2.30 +/- 2.68 vs. 1.33 +/- 1.82, p < .05). HR and HF (n.u.) were also higher in younger than older women. LF/HF was lower in women than men. Percentile curves showed almost all HRV indices decreasing with aging. The availability of short-term, resting-state HRV reference values in a large sample of healthy and nonmedicated participants from 35-74 years will provide a valuable tool for researchers, clinicians, and those in the quantified-self community.