The parasympathetic nervous system plays a major role in the pathophysiology of many cardiovascular disease, particularly in modulating myocardial electrical stability. Measurements of heart rate variability have been widely used to assess parasympathetic activity. The reproducibility of measurements obtained from 24-hour ambulatory electrocardiograms has not been well documented. We have developed a technique for measuring parasympathetic activity from clinical quality 24-hour ambulatory electrocardiograms by counting beat-to-beat increases in RR interval that are > 50 ms. To determine the reproducibility and sensitivity of our technique, we analyzed repeated 24-hour electrocardiograms of 173 subjects (19 normal subjects, 67 patients with ischemic heart disease, and 87 diabetics) followed up over periods of 2 to 16 weeks. In all subject groups, mean values for repeated measurements were virtually identical. Measurements were stable in all 3 groups throughout the course of the study, as assessed by intraclass correlation coefficients. This technique is sensitive enough to detect relatively small changes in parasympathetic activity in subjects, as demonstrated by the calculated Bland and Altman coefficients of repeatability. Reproducibility and sensitivity of our technique are particularly good in normal subjects and in patients with ischemic heart disease. The results obtained with this technique imply that other related measurements of parasympathetic activity will show similar excellent short- and long-term reproducibility and sensitivity.