Significance. Quantifying human milk composition is important for daily nutritional management in neonatal intensive cares worldwide. Photonic solutions based on visible light can potentially aid in this analysis, as energy content of human milk depends largely on fat content, and the optical scattering properties of human milk predominantly depend on the size and concentration of fat globules. However, it is expected that human milk scattering changes upon homogenization, routinely done before analysis, which may affect fat globule size.

Aim. The first aim of this study was to investigate how the most common homogenization methods (gently inverting by hand, vortexing, and sonication) affect the optical properties of human milk. The second aim was to estimate the scattering contribution of casein micelles, the second most dominant scatterers in human milk.

Approach. We combined diffuse reflectance spectroscopy with spectroscopic optical coherence tomography to measure the scattering coefficient μ_s, reduced scattering coefficient μs′, and anisotropy g between 450 and 600 nm.

Results. Sonication induced the strongest changes in μ_s, μs′, and g compared to the gently inverted samples (203%, 202%, and 7%, respectively, at 550 nm), but also vortexing changed μs′ with 20%. Although casein micelles only showed a modest contribution to μ_s and g at 550 nm (7% and 1%, respectively), their contribution to μs′ was 29%.

Conclusions. The scattering properties of human milk strongly depend on the homogenization method that is employed, and gentle inversion should be the preferred method. The contribution of casein micelles was relatively small for μ_s and g but considerably larger for μs′.