Diffuse optical tomography (DOT) is a noninvasive, nonionizing imaging modality that uses near-infrared light to visualize optically relevant chromophores. A recently developed dynamic DOT imaging system enables the study of hemodynamic effects in the breast during a breath-hold. Dynamic DOT imaging was performed in a total of 21 subjects (age ) including 3 healthy subjects and 18 subjects with benign () and malignant () masses. Three-dimensional time-series images of the percentage change in oxygenated and deoxygenated hemoglobin concentrations ( and [Hb]) from baseline are obtained over the course of a breath-hold. At a time point of 15 s following the end of the breath-hold, [Hb] in healthy breasts has returned to near-baseline values (), while tumor-bearing breasts have increased levels of [Hb] (, ). Further, healthy subjects have a higher correlation between the breasts over the course of the breath-hold as compared with the subjects with breast cancer (healthy: ; benign: ; malignant: , ). Therefore this study shows that dynamic features extracted from DOT measurements can differentiate healthy and diseased breast tissues. These features provide a physiologic method for identifying breast cancer without the need for ionizing radiation.