Although many laboratory studies have demonstrated changes in cardiac autonomic control during psychological stress, few have attempted to demonstrate this effect in ambulatory subjects. To address this issue, 24-h electrocardiographic recordings of 33 healthy subjects were analyzed for RR interval and heart period variability (HPV) responses associated with periodic diary entries measuring physical position, negative effect, and time of day. A total of 362 diary entries were made during the 24-h sessions, each in response to a device which signaled on an average of once per hour. HPV was analyzed in the frequency domain, yielding estimates of spectral power in low (LF) and high (HF) frequency bands, as well as the LF/HF ratio. Because of the high correlations of the measures of negative affect (alpha = 0.91), they were combined to create a single index of stress. Multivariate analysis was used to assess the effect of individual subject differences, physical position, and stress on RR interval and HPV. Results revealed significant effects of individual differences, stress, and physical position on RR interval, with increases in stress associated with decreases in RR interval as expected. HF power was significantly lower and the LF/HF ratio significantly higher in the standing compared with the sitting position. Psychological stress was significantly associated with an increase in the LF/HF ratio, suggesting increases in the relative predominance of sympathetic nervous system activity during stressful periods of the day. Overall, these findings suggest that in ambulatory normal subjects, cardiac autonomic control varies throughout the day as a function of self-reported stress.