In general, the application of HPT processing at 300, 600, and 900 MPa combined with 50, 65, and 80 °C in human milk, enhanced the levels of derived compounds from lipid oxidation and Maillard reaction such as aldehydes, ketones, furans and pyrans, and alcohols. In contrast, it reduced the content of aliphatic hydrocarbons and did not affect the levels of carboxylic acids. Negative odor attributes in human milk may be derived from these variations. Taking into account that changes in the volatile profile reflect modifications of the general composition of milk, we could assume that the HPT conditions utilized in the present study may not be adequate to process human milk. Nevertheless, we consider particularly relevant to assay different combinations of pressures and temperatures at less intense conditions to conclude the efficacy of the HPT technology to preserve milk quality in human milk banks.