Simple SummaryIf measured in the high-frequency (HF) domain, heart rate variability (HRV) reflects the tonic, inhibitory control by the parasympathetic nervous system (PNS) to decelerate the heart rhythm. Several studies have examined changes in HF-HRV variability across the menstrual cycle; however, they did not implement the latest technological and statistical approaches. The evaluation of HF-HRV across the menstrual cycle is important because low HF-HRV variability is observed in numerous conditions, including heart disease and depression. Moreover, if a change in HF-HRV is observed around ovulation, it may be used to time conception. This study found that HF-HRV significantly decreases around ovulation relative to all other times of the menstrual cycle. This finding enhances our understanding of human biology. Future studies may examine whether the decrease in HF-HRV is a normal process that may be utilized in the future to enhance fertility rates, or whether it represents a biological process that may predispose some women to disease development.Abstract(1) Background: High-frequency heart rate variability (HF-HRV) is an essential ultradian rhythm that reflects the activity of the PNS to decelerate the heart. It is unknown how HF-HRV varies across the menstrual cycle (MC), and whether progesterone mediates this potential variation. (2) Methods: We enrolled 33 women in the study to attend eight clinic visits across the MC, during which we measured their resting HF-HRV and collected samples for the analysis of luteinizing hormone (LH) and progesterone. We realigned the study data according to the serum LH surge to the early follicular, mid-follicular, periovulatory, early luteal, mid-luteal and late luteal subphases. (3) Results: Pairwise comparisons between all the subphases showed significant differences between the early follicular and periovulatory subphases (β = 0.9302;p≤ 0.001) and between the periovulatory and early luteal subphases (β = −0.6955;p≤ 0.05). Progesterone was positively associated with HF-HRV in the early follicular subphase but not the periovulatory subphase (p≤ 0.05). (4) Conclusions: The present study shows a significant drop in HF-HRV in the anticipation of ovulation. Further research in this area is critical given the marked cardiovascular disease mortality in women.