To test the hypothesis that certain lifestyles may affect cardiovascular regulatory mechanisms, heart rate variability (HRV) among three age-matched groups with different lifestyles (smoking, sedentary and aerobically fit) were compared. Heart rate variability was defined as the difference in heart rate during inhalation vs. exhalation. Heart rate was obtained from normal RR intervals, using a continuous electrocardiogram recording, while subjects were seated and breathing at an augmented tidal volume, and also while subjects were standing and breathing at normal tidal volumes. In the physically active group, heart rate variability was significantly elevated at rest as well as during some of the autonomic tests, when compared to the sedentary and smoker groups (p < 0.05). A hypothesis to explain this finding is that smoking or a sedentary lifestyle reduces vagal tone, whereas a physically active lifestyle, resulting in enhanced aerobic fitness, increases vagal tone. These findings may have cardiovascular health implications.