When a mother’s milk is unavailable, the best alternative is donor milk (DM). Milk delivered to Human Milk Banks should be pasteurized in order to inactivate the microbial agents that may be present. Currently, pasteurization, performed at 62.5 °C for 30 min (Holder Pasteurization, HoP), is recommended for this purpose in international guidelines. Several studies have been performed to investigate the effects of HoP on the properties of DM. The present paper has the aim of reviewing the published papers on this topic, and to provide a comparison of the reported variations of biologically-active DM components before and after HoP. This review was performed by searching the MEDLINE, EMBASE, CINHAL and Cochrane Library databases. Studies that clearly identified the HoP parameters and compared the same DM samples, before and after pasteurization, were focused on. A total of 44 articles satisfied the above criteria, and were therefore selected. The findings from the literature report variable results. A possible explanation for this may be the heterogeneity of the test protocols that were applied. Moreover, the present review spans more than five decades, and modern pasteurizers may be able to modify the degradation kinetics for heat-sensitive substances, compared to older ones. Overall, the data indicate that HoP affects several milk components, although it is difficult to quantify the degradation degree. However, clinical practices demonstrate that many beneficial properties of DM still persist after HoP.