Heart rate variability (HRV) and phase-rectified signal averaging (PRSA) estimates of heart rate dynamics are diminished in older people compared with younger people. However, it is not fully elucidated whether these differences are related to age per se or to the concomitant influence of aerobic fitness. Aerobic fitness (peak oxygen uptake, gas exchange threshold, oxygen uptake kinetics, exercise economy) was assessed in 70 healthy adults (41 male) aged 18-57 years. Participants also underwent a 24 h, ambulatory ECG for the derivation of HRV and PRSA variables. HRV was most sensitive to age and aerobic fitness when measured during the morning period (6 am-12 pm). HRV and PRSA were both diminished with age and were higher in aerobically superior participants. The decline in HRV with age was predominantly attributable to age itself (33%), with aerobic fitness representing an additional modulating factor. The present study also provides tentative evidence that assessment of the influence of aerobic fitness should not rely on [Formula: see text]O(2peak) alone. These findings demonstrate that age per se is an important factor in determining HRV. However, given the clinical importance of diminished HRV and the immutable nature of aging, the potential significance of physical activity/training to enhance cardiac regulatory function should not be underestimated.