Intralipid has become an extensively studied and widely used reference and calibration phantom for diffuse optical imaging technologies. In this study we call attention to the layering properties of Intralipid emulsions, which are commonly assumed to have homogeneous optical properties. By measurement of spatial frequency domain reflectance in combination with an analytical solution of the radiative transfer equation for two-layered media, we make quantitative investigations on the formation of a surface layer on different dilutions of Intralipid. Our findings are verified by an independent spatially resolved reflectance setup giving evidence of a time dependent, thin and highly scattering surface layer on top of Intralipid-water emulsions. This layer should be considered when using Intralipid as an optical calibration or reference phantom.