We aimed to evaluate if human milk-based fortifier (HMBF) affects human milk fat globule (MFG) size less than cow milk-based fortifier (CMBF), which may impact overall infant feeding tolerance. Measurements of donated human milk were performed before fortification as well as at 1 hour, 24 hours, and 48 hours after fortification with CMBF or HMBF. MFG size in each sample of fortified milk was measured by laser light scattering. MFG size in the fortified milks increased gradually over time. At 24 and 48 hours after fortification, MFG size in the milk with CMBF was larger than that in the milk with HMBF (4.8 ± 0.5 vs 4.3 ± 0.3 μm, p<0.01, 5.1 ± 0.7 vs 4.5 ± 0.4 μm, p = 0.03, respectively). HMBF is associated with less alteration of MFG size than CMBF. This may have an impact on feeding tolerance of very preterm infants.