Background <p>Early and increased exposure to human milk combined with minimal exposure to parenteral nutrition could reduce hospitalisation costs, improve postnatal growth, and influence the diversity of the gut microbiome.</p> Methods <p>Faecal samples from very preterm infants randomised to receive either 60 to 80 ml/kg/day (intervention group) or 20 to 30 ml/kg/day (control group) of human milk within the first 36 h after birth were collected at approximately postnatal day 14. After trial completion, samples were analysed by 16S rRNA gene sequencing to determine early differences in the gut microbiome between the randomisation groups using adjusted models.</p> Results <p>We analysed faecal samples from 95 infants with a median gestational age of 31 weeks (mean birthweight: 1487 g). In adjusted analyses, taxonomic richness and Shannon alpha diversity were not significantly higher in the intervention group. No significant differences in microbial diversity composition between samples (i.e., beta diversity) were found. Four distinctive de novo community clusters were identified during the trial, but they did not differ according to randomisation groups.</p> Conclusion <p>Early and increased exposure to human milk shortly after birth does not appear to increase the richness and diversity of the gut microbiome by postnatal day 14 in very preterm infants.</p> Trial registration <p>ClinicalTrials.gov identifier: NCT04337710</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>In very preterm infants, early and increased exposure to human milk and its bioactive components did not alter gut microbiome richness or diversity by postnatal day 14.</p> </ItemContent> <ItemContent> <p>Randomisation strengthens microbiome analyses by limiting confounding in human milk feeding trials.</p> </ItemContent> </UnorderedList></p>