The objective of the present study was to investigate in healthy young women the fluctuations in serum concentration of human epididymal secretory protein human epididymis-specific protein 4 (HE4) and CA125 during the phases of the menstrual cycle and the correlation between HE4 and CA125 values and age. Forty women with regular menstrual cycles were included in the study. Pelvic and transvaginal ultrasound were performed in order to exclude ovarian pathologies. Blood samples were collected at follicular (FP), ovulatory (OP), and luteal (LP) phases of the hormonal cycle. The values of HE4 (expressed as picomoles per liter) observed were (mean ± SEM) 39.1 ± 1.1 (FP), 45.3 ± 1.19 (OP), and 42.0 ± 1.3 (LP). The difference between FP and OP was statistically significant (p = 0.0002). By contrast, serum CA125 levels (expressed as units per milliliter) were 14.35 ± 0.66 (FP), 13.15 ± 0.54 (OP), and 13.70 ± 0.54 (LP), respectively. The levels of HE4 observed in serum samples of women below 35 years were 37.5 ± 1.28 in the FP, 46.6 ± 1.4 in the OP, and 42.8 ± 1.49 in the LP. In this group, a statistically significant difference was observed in the FP compared with the OP (p < 0.0001), whereas no statistically significant difference was observed during the different hormonal phases in the group of women over 35. In conclusion, the correct interpretation of laboratory data is essential to define a threshold of normality, and for what concerns HE4 levels, the menstrual cycle phase-dependent variability appears indicated in the interpretation of the results.