This study was designed to assess the effects of age and physical fitness on vagal modulation of heart rate (HR) during exercise by analyzing the instantaneous R-R interval variability from Poincaré plots (SD1) at rest and at different phases of a bicycle exercise test in a population of healthy males. SD1 normalized for the average R-R interval (SD1n), a measure of vagal activity, was compared at rest and during exercise among subjects of ages 24-34 (young, n = 25), 35-46 (middle-aged, n = 30), and 47-64 yr (old, n = 25) matched for peak O2 consumption (VO2 peak) and among subjects with VO2 peak of 28-37 (poor, n = 25), 38-45 (average, n = 36), and 46-60 ml.kg-1.min-1 (good, n = 25) matched for age. SD1n was higher at rest in the young subjects than in the middle-aged or old subjects (39 +/- 14, 27 +/- 16, and 21 +/- 8, respectively; P < 0.001), but the age-related differences in SD1n were smaller during exercise [e.g., 11 +/- 5, 9 +/- 5, and 8 +/- 4 at the level of 100 W; P = not significant (NS)]. The age-matched subjects with good, average, and poor VO2 peak showed no difference in SD1n at rest (32 +/- 17, 28 +/- 13, and 26 +/- 11, respectively; P = NS), but SD1n differed significantly among the groups from a low to a moderate exercise intensity level (e.g., 13 +/- 6, 10 +/- 5, and 6 +/- 3 for good, average, and poor fitness groups, respectively; P < 0.001, 100 W). These data show that poor physical fitness is associated with an impairment of cardiac vagal function during exercise, whereas aging itself results in more evident impairment of vagal function at rest.