This study investigated menstrual cycle phase differences in heart rate (HR) and RR interval variability (RRV) in 49 healthy, premenopausal, eumenorrheic women (age 30.2+/-6.2 years). HR and RRV were computed from ambulatory 24-h electrocardiogram, collected for up to 6 days, with at least 1 day each during early to midfollicular and midluteal menstrual phases. Phase effects on HR and RRV were assessed using linear mixed effects models with a random intercept to account for the correlation of observations within each subject as well as intrasubject variation. During follicular phase monitoring, women had significantly lower average HR (-2.33 bpm), and higher standard deviation, the root mean squared successive difference, and high frequency (0.04-0.15 Hz) and low frequency (0.15-0.40 Hz) RRV than during the luteal phase. These results provide strong support for the influence of menstrual phase on cardiac autonomic regulation in premenopausal women.