The purpose of the present study was to measure changes in blood volume and the oxygenation state of breast tissue during breastfeeding with the use of near-infrared time-resolved spectroscopy (TRS) as a noninvasive method. Forty breastfeeding mothers were divided into 2 groups in which measurements were made in the contralateral breast (group A) and in the ipsilateral breast (group B). The tissue concentrations of oxyhemoglobin, deoxyhemoglobin, and total hemoglobin and the hemoglobin oxygen saturation were measured in the breast by using TRS during a breastfeed. In both groups A and B, the values of all parameters decreased significantly after the start of breastfeeding compared with the pre-breastfeeding values. The values of all parameters fluctuated cyclically after the beginning of the breastfeed. Time-resolved spectroscopy is an effective noninvasive method for investigating the hemodynamics of the breast during breastfeeding.