BackgroundHuman milk (HM) for premature infants is frequently Holder pasteurized (heated at 62.5 ± 0.5°C for 30 min) despite its detrimental effects on heat-sensitive milk components. This tolerated compromise ensures HM's microbial safety while less detrimental methods like short-time HM treatments (HTST) are still being evaluated. Dry-tempering devices (DT-HoP) were recently introduced in clinical practice due to hygienic concerns about water-based Holder pasteurizers (WB-HoP). Evidence on the impact of such dry-tempering devices on HM quality is lacking. The aim of this study was to compare protein retention rates after DT-HoP, WB-HoP and HTST.MethodsWe colorimetrically determined alkaline phosphatase activity (ALP), concentrations of secretory immunoglobulin A (sIgA), and lactoferrin (LF) before and after DT-HoP, WB-HoP and HTST.ResultsALP was below the detection limit after HoP, but retained 52.8 ± 13% activity after HTST (p < 0.01). Secretory IgA (WB-HoP = 73.2 ± 13.5% vs. DT-HoP = 57 ± 14%, p = 0.0018) and LF retention (WB-HoP=47 ± 40% vs. DT-HoP=25 ± 8%, p = 0.07) differed between the two HoP modes. Again, retention was better maintained after HTST compared to HoP (80.4 ± 23% sIgA and 70 ± 42% LF concentration, all p < 0.01).ConclusionDry-tempering milk lowers even further the quality of HM when performing HoP compared to water-bath pasteurization, while HTST warrants continued evaluation for clinical application.