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.