The time and frequency domain components of heart rate variability have been used to assess prognosis in patients with different types of heart disease. However, the effect of habitual exercise, which influences baseline parasympathetic tone, on heart rate variability has not been fully evaluated. To determine the effect of chronic exercise on heart rate variability, we studied 12 athletes and 18 control subjects. Time domain and frequency domain analysis was performed on 15-minute resting heart rate acquisitions. Athletes had evidence of increased vagal activity in the time domain compared with control subjects (eg, increased standard deviation of R-R intervals) but showed evidence of decreased power in variables reflecting vagal activity in the frequency domain (eg, total power and high-frequency power). Of note, there was good correlation between time and frequency domain variables, which reflected parasympathetic tone in the control group that was not seen in athletes. These data suggest that frequency domain analysis of heart rate variability may not be an accurate indicator of cardiac vagal tone in chronically trained endurance athletes and activity level may have to be considered when using heart rate variability to carry out prognostic stratification in patients with heart disease.