PURPOSE: To investigate the inter-day reliability of time-domain, frequency-domain, and nonlinear HRV metrics in healthy highly active younger and older adults. The study also assessed the effect of age on the HRV metrics. METHODS: Forty-four older adults (34 M, 10F; 59 +/- 5 years; [Formula: see text] = 40.9 +/- 7.6 ml kg(-1) min(-1)) and twenty-two younger adults (16 M, 6F; 22 +/- 4 years; [Formula: see text] = 47.2 +/- 12.8 ml kg(-1) min(-1)) attended the laboratory. Visit one assessed aerobic fitness through an exercise test. In visits two and three, participants completed a 30-min supine RR interval measurement to derive the HRV metrics. RESULTS: The younger group (YG) and older group (OG) demonstrated poor to good day-to-day relative and absolute reliability for all HRV metrics (OG, ICCs = 0.33 to 0.69 and between day CVs = 3.8 to 29.2%; YG, ICCs = 0.37 to 0.93 and between day CVs = 3.5 to 36.5%). There was a significant reduction in ApEn (P < 0.001), SampEn (P = 0.031), RMSSD (P < 0.001), SDNN (P < 0.001), LF power (P < 0.001) and HF power (P < 0.001), HRV metrics with ageing. There was no significant effect of age the complexity metrics DFA alpha1 (P = 0.107), alpha2 (P = 0.147) and CI-8 (P = 0.493). CONCLUSION: HRV metrics are reproducible between days in both healthy highly active younger and older adults. There is a decline in linear and nonlinear HRV metrics with age, albeit there being no age-related change in the nonlinear metrics, DFA alpha1, alpha2 and CI-8.