Abstract Objective To determine, by the creamatocrit measurement, the effect on the fat content of raw and pasteurized donor milk of freezing during 3 months at −20 °C. Methods The evolution of the creamatocrit measurement (following Lucas technique) on frozen (−20 °C), raw and pasteurized human milk, was analyzed during 3 months. Results The fat content of raw milk (n=44) was 3.19 g/dl at the beginning and 2.86 g/dl after 3 months frozen (p=0.02). In pasteurized milk (n=36) fat content at the first determination was 2.59 g/dl and 2.20 g/dl after 1 month frozen (p=0.01). Afterwards there were no significant changes up to 3 months frozen. Variability was observed in the intermediate values. Conclusions A reduction on the fat content measurement of raw and pasteurized donor human milk after freezing was observed. Freezing does not inactivate the milk lipase but does destroy the fat globule. Creamatocrit measurement may not be the best method to determine the fat content of processed human milk.