Breastfeeding plays a crucial role in public health, but relatively few imaging and sensing technologies are employed to study human lactation physiology. As a consequence, many breastfeeding problems are not well understood. We hypothesize that diffuse optical spectroscopic imaging (DOSI) can potentially reveal important physiological parameters that help to define milk synthesis and secretion: glandular tissue content, hemodynamics and milk ejection. The aim of this study is to investigate the sensitivity of DOSI to these physiological parameters in (i) a case study (1 subject) on mammary involution of the lactating breast to its pre-pregnant state and (ii) a pilot study during milk extraction with a breast pump (4 lactating subjects, 5 non-lactating subjects). For the case study, the measured changes in the DOSI parameters (water, lipid, hemoglobin concentration) were consistent with the gradual replacement of fibro-glandular tissue by adipose tissue and vascular regression during mammary involution. For the pilot study, the measured changes in the DOSI parameters correlated with the extracted milk volume and occurrence of the milk ejection reflex. In conclusion, DOSI is sensitive to human lactation physiology, which can potentially aid to obtaining an in-depth understanding on the origin and treatment of breastfeeding problems.