Several papers show that NIR technique is valuable auxiliary tool to aid the operator through the cheese-making process management. This capability relies mainly on the variations of the light scattering induced by the formation of the clot after the rennet addition to the milk. Light scattering and spectral baseline are also affected by fat globules that can interfere with the scope of the measurements: the aim of this study was to verify the actual influence of milk fat content on the spectral response, when monitoring the milk coagulation process, using an aquaphotomics approach. This study was conducted through an experimental design, in which different levels of fat and amounts of rennet were considered. Three different doses of calf rennet were added to a reconstituted powder cow milk previously acidified with natural starter whey. The NIR spectra of the milk with rennet, were collected in reflectance mode, every 5s during the coagulation, with a MicroNIR spectrometer, (VIAVI) at constant temperature. Evaluating the linear model calculated from the full factorial experimental design for predicting the clotting time it was verified that the fat content and the interaction “fat x calf rennet content” were not significant. Aquagrams were developed considering the absorbance values of wavelengths, in the first overtone of the O–H stretching band, which resulted significant in the exploratory PCA of the spectra. Linear models with interaction were calculated separately considering the spectra collected at each two-minute interval of the coagulation process, using the normalized absorbances of the most significant wavelengths as responses. It was verified that fat content has a certain influence on the spectral response, in the range of the first overtone, particularly in the early stages of the coagulation process.