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The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m 2 per buffalo and group S15 had a space allocation of 15 m 2 per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl-l-carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl-l-carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 mM Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1β, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)