Information about lipid oxidation in fresh and stored human milk compared with infant formulas is scarce. We aimed to assess n-6 and n-3 PUFA oxidation in these milks by measuring the 4-hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE) content. Human milk samples (n = 4), obtained from volunteer mothers, were analyzed fresh and after 1 wk at 4°C or 24 h at 18°C. Vitamin E and malondialdehyde (MDA) were measured by HPLC and fatty acid profile by GC. The 4-HHE and 4-HNE contents were measured by GC-MS. Infant formulas (n = 10) were tested; their fat droplet size was measured by laser light scattering and observed by confocal laser scanning microscopy. Human milk samples contained 31.0 ± 6.3 g/L of lipids and 1.14 ± 0.26 mg/L of vitamin E. Fat droplets were smaller in infant formulas than reported in human milk. The (4-HHE/n-3 PUFA) ratio was 0.19 ± 0.01 μg/g in fresh human milk (unchanged after storage) versus 3.6 ± 3.1 μg/g in dissolved powder formulas and 4.3 ± 3.8 μg/g in liquid formula. (4-HNE/n-6 PUFA) was 0.004 ± 0.000 μg/g in fresh milk (0.03 ± 0.01 μg/g after storage) versus 1.1 ± 1.0 μg/g in dissolved powder formulas and 0.2 ± 0.3 μg/g in liquid formula. Infant formulas also contained more MDA than human milk. n-3 PUFA were more prone to oxidation than n-6 PUFA. Whether threshold levels of 4-HHE and 4-HNE would be of health concern should be elucidated.