Background <p>Human milk (HM) is the first form of communication between mothers and newborns and it is implicated in the infant growth and protection. We recently showed a functional characterization of HM, unmasking the molecular mechanisms related to EVs signaling and its functional role in prematurity. In that study, we identified the need to establish and optimize a standard isolation protocol for human milk extracellular vesicles (mEVs).</p> Methods <p>Four mEVs isolation methods were compared: ultracentrifugation (UC), size exclusion chromatography (SEC), immunoprecipitation with tetraspanin CD9 (IP_CD9) and ExoGAG. Three pools of human milk (each composed of samples from ten donor mothers) were used and isolation of mEVs was performed starting from the same volume for each method. The proteomic, transcriptomic and glycomic composition of the extracellular vesicles obtained after isolation was then analyzed for each method. The sensitivity, specificity and quality of the results were also determined. A comparative analysis of results common across all isolation methods was performed to identify potential signaling pathways associated with mEVs.</p> Results <p>ExoGAG and UC proved to be the most efficient of the four techniques compared for mEVs isolation. However, ExoGAG compared to UC provided a higher concentration of total and vesicle-related proteins and peptides and a higher glycoprotein count keeping all the glycan subgroups. Despite ExoGAG and UC show similar vesicle profiles in terms of size, concentration, tetraspanin subpopulations and EVs markers, ExoGAG was the most efficient technique in terms of accuracy, consistency and reproducibility for omics studies. Furthermore, results allowed us to identify that mEVs components are involved in the signaling pathways of infant biological development, immune system maturation and protein metabolism.</p> Conclusions <p>This study establishes UC and ExoGAG as reliable methods for mEVs isolation and describes its protocol, being ExoGAG the most efficient. Also, the omics analysis show that biomolecules conforming those mEVs are linked to the defense system against external agents (specific role in the immune system pathway) and in the correct establishment of the neural structure (developmental pathway), while providing all the nutritional requirements for the correct growth of the newborn (metabolic pathway).</p> Graphical Abstract <p></p>