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Mastodynia is correlated with the menstrual cycle. Using frequency-domain near-infrared spectroscopy (FD-NIRS), we investigated changes in breast perfusion in women who were or were not using hormonal contraception. Healthy volunteers, on or not on hormonal contraception, were examined. Optical properties were measured in all quadrants of both breasts, and physiological parameters were calculated. Measurements were repeated every other day during one complete menstrual cycle. Measurements were comparable in all quadrants. Data remained unchanged during the entire cycle in patients using hormonal contraception. However, a biphasic variation of deoxyhemoglobin, oxyhemoglobin, total hemoglobin (tHb), and water content (H2O) was observed in women not using contraception. tHb and H2O distinctly increased during the ovulation period and remained elevated throughout the luteal phase. It was concluded that FD-NIRS allows accurate measurement of optical properties of human breasts. As opposed to the menstrual cycles of persons using oral contraception, spontaneous menstrual cycles exhibit biphasic variations of tissue perfusion parameters. These findings are important for the investigation of mastodynia.