The in vitro bioactivity of 5 kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes were compared. Reverse phase ultra-performance liquid chromatography and gel permeation chromatography showed that the permeates had different physicochemical properties (molecular mass and degree of hydrolysis). The Flavourzyme (R) permeate had the highest activity in the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Cellular antioxidant and immunomodulatory assays showed that none of the permeates exhibited in vitro antioxidant activity, while all permeates significantly (P < 0.05) decreased interleukin-6 (IL-6) production in ConA-stimulated Jurkat T cells at 0.50% (w/v) and LPS-stimulated RAW264.7 cells at 0.05 and 0.50% (w/v). Three permeates, obtained using Flavourzyme (R), Flavorpro Whey and trypsin, also significantly (P < 0.05) decreased IL-1 beta production at 0.05% (w/v) in RAW264.7 cells. Western blot analysis showed that all permeates significantly decreased the expression of the NF-kappa B subunit, p65, in RAW264.7 cells indicating that anti-inflammatory activity may be associated with this pathway. (C) 2019 Elsevier Ltd. All rights reserved.