Background: Holder pasteurization is commonly used in milk banks. We previously reported that the pattern of temperature and time may be different according to the pasteurizer used. Aim: The aim of our study was to assess the variances in pasteurization using two different devices: a standard pasteurizer (Past STD) and an optimized pasteurizer (Past OPTI). Methods: lmmunoglobulin A (IgA), lactoferrin (LF), and lysozyme (LZ) content were assessed before and after pasteurization of 24 donor human milk samples. The impact of the pasteurization device was evaluated by testing 50- to 200-mL samples. Results: Mean temperature and duration of the plateau were 1.5 degrees C lower and 11 min shorter, respectively, with Past OPTI vs. Past STD. The loss of IgA, LF, and 1.2 was 17.6, 5.6, and 9.8% lower, respectively, with Past OPTI than with Past STD. Conclusions: Accurate control of temperature enabled better preservation of IgA, LF, and LZ in donor milk. Holder pasteurization should be optimized, and new techniques proposed to treat donor milk should be compared with Holder pasteurization performed with a well-controlled device under realistic conditions.