Clinical near-infrared (NIR) imaging is a diagnostic tool to quantify hemoglobin concentration and oxygen saturation within tissue. In order to remove system offsets and correct data acquired from the breast during clinical examination, a homogenous tissue-simulating phantom is routinely employed for system calibration. In this study, both hard resin and soft silicon-based materials were used to make calibration phantoms with different physical sizes, shapes, elasticity, and optical properties. Using these calibration phantoms, the imaging performance of two different fiber optic configurations was tested. Images from a patient with a biopsy-confirmed ductal carcinoma tumor were also used to determine the effect of calibration object choice upon real tissue image reconstructions. Softer silicon-based calibration phantoms were found to mimic the elastic properties of human breast tissue quite well with an elastic modulus that could be tailored between 100 and 230 KPa. These values agree with estimates of breast tissue elasticity and we have found that these phantoms provide an optimal model for mimicking breast tissue, while having homogeneous properties, which are ideal for calibration and validation of the system. In this study, the performance of the imaging system with colocalized source and detector optical fibers (transceiver system) yielded better image quality than the system with separated-source detector fibers. In vivo imaging of the ductal carcinoma revealed increased total hemoglobin within the tumor. © 2003 SPIE and IS&T.