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It is known that breastfeeding protects the infant from enteric and respiratory infections; however, the antiviral properties of human milk against enteric and respiratory viruses are largely unexplored. To explore the antiviral activity of human preterm colostrum against rotavirus and respiratory syncytial virus and to assess whether the derived extracellular vesicle contribute to this activity. We used a cross-sectional, prospective two-group non-experimental design. Colostra were collected from mothers of preterm newborns ( = 1 and e