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We evaluated the effect of in vitro digested milk on mature adipocytes 3T3-L1, paying particular attention to its fatty acid composition, and comparing human (HM), donkey (DM), bovine (BM), ovine (OM), caprine (CM) and formula (FM) milk. Cellular viability, apoptosis, oxidative response and gene expression levels of NF-κB p65, HMGB1, SREBP-1c and FAS were evaluated. Digested milk treatments significantly reduced 3T3-L1 mature adipocytes viability and caspase activity compared with control group, but no significant differences were observed among different sources of digested milk. In all digested milk samples, ROS level was higher than the control, however, the digested human and formula milk showed lower levels of ROS than DM, BM, OM and CM samples. Lower capacity of HM and FM to induce oxidative stress in mature adipocytes was ascribed to the peculiar free fatty acids profile of digested milk samples. All milk treatments elicited a significant over-expression of NF-κB p65 in 3T3-L1 adipocytes compared to the control; the lowest gene expression was found in HM, BM, OM and CM, the highest in FM and an intermediate behavior was shown in DM. All digested milk treatments influenced the gene expression of SRBP-1c with FM and HM showing the highest levels. For FAS expression, BM showed the highest level, OM and CM intermediate and FM, HM and DM the lowest levels, however HM and DM had comparable levels to the control.