With human milk being the most important source of infant nutrition, the protection and support of breastfeeding are essential from a global health perspective. Nevertheless, relatively few objective methods are available to investigate human milk composition and lactation physiology when a mother experiences breastfeeding problems. We argue that optics and photonics offer promising opportunities for this purpose. Any research activity within this new application field starts with a thorough understanding on how light interacts with human milk. Therefore, the aim of this study was to investigate the full set of optical properties for human milk and the biological variability therein. Using a novel approach that combines spatially resolved diffuse reflectance spectroscopy (SR-DRS) and spectroscopic optical coherence tomography (sOCT) between 450 and 650 nm, we quantified the absorption coefficient µa, scattering coefficient µs, reduced scattering coefficient µs’, anisotropy g and backscattering coefficient µb,NA of mature human milk from 14 participants released at different stages during a breastfeed (foremilk, bulk milk and hindmilk). Significant correlations were found between µa, µs, µs’ and µb,NA and the biochemically determined fat concentration per sample (Rs = 0.38, Rs = 0.77, Rs = 0.80, Rs = 0.44 respectively). We explained the observed variations in the optical properties of human milk using Mie theory and the biological variability in both the concentration and size distribution of milk fat globules. In conclusion, we have provided a full set of optical properties for human milk, which can hopefully serve as a starting point for future biophotonic studies on human milk and the milk containing lactating breast.