Purpose: Heart rate variability (HRV), a characteristic that is potentially increased by physical activity, has been associated with incidence of cardiac events and total mortality. Since the incidence of cardiac events among older people is high and their physical activity levels and HRV are generally low, it is important to investigate whether regular physical activity can modify HRV in this age group. The purpose of the study was to investigate the effect of regular physical activity on HRV in older men and women. Methods: In a randomized controlled trial, the effect of six months' training on HRV was investigated in a group of 51 older men and women (67.0 ± 5.1 yr). The training group gathered three times per week for 45 min supervised training. Results: At the end of the intervention period, HRV was higher primarily during the day. During daytime, the SD of all normal intervals (+ 6%) as well as the low frequency component (+ 15%) and the very low frequency component (+ 10%) of HRV were significantly increased (P < 0.05) as compared with the control group. Effects of training were most pronounced in subjects inactive in sports at baseline. Conclusion: This study demonstrates that regular physical activity increases HRV (specifically in the very low and low frequency components) in older subjects. Hence, in older subjects, physical training may be an effective means to modify positively a factor that is associated with increased incidence of cardiac events.