Introduction. In previous studies, it has been observed that the concentration of some components of breast milk such as proteins, carbohydrates, lipids among others vary throughout the day. In the same way, it has been observed that bacteria diversity undergoes changes at the same time interval. In human milk there are also beneficial bacteria from the mother that are transferred to the newborn by breastfeeding, being the main source of colonizing bacteria of the newborn's gastrointestinal tract. Many of these bacterial communities display oscillations throughout the day, contributing to homeostasis and play an important role in various physiological processes. It is reported that the healthy human microbiome is regulated by the circadian cycle, where fluctuations in abundance of the bacterial community occur, in addition to a relationship in feeding times and sleep cycles of the mother. The aim of this work was to identify possible fluctuations and variations in bacterial diversity and composition on human milk throughout the day. Methods Human milk samples were collected during 5 consecutive days from Monday to Friday at three different times, approximately every 8 hours: Morning (7:00 am), Afternoon (3:00 pm), and night (10:00 pm). Samples were manually collected in aseptic conditions and immediately freeze at -20° C. Bacterial DNA was extracted using the Favorgen stool kit. The diversity was characterized by high-throughput DNA sequencing of V3-16S rDNA libraries. The sequencing data were analyzed using the QIIME pipeline and taxonomy was assigned by comparison of the sequences against the Greengenes reference database. Alpha diversity was determined using Phyloseq and ggplot2 packages in the R environment (v3.4.4). For beta diversity, the dissimilarity was estimated using unweighted Bray Curtis analysis. The linear discriminant analysis effect size program (LEfSe v1.0) was used to detect significant differences in the relative abundances of bacterial taxa. Results. The human milk microbiota shows changes in the bacterial composition between morning and night; also, we observed significant changes in the Shannon diversity index between both groups. On the other hand, no differences were observed in the bacterial composition or alfa diversity in the afternoon samples compared with the morning and night samples. For beta diversity analysis, no clustering was observed among all the samples. As previously has been reported, Lactobacillus was found more enriched in the night milk of samples with statistical significance; same was observed for environmental proteobacteria and actinobacteria. The family Micrococcaceae was detected as more abundant in samples collected in the morning. Conclusions. As for other human milk components, the human milk microbiota seems to be affected by the daytime changes. The results presented in this work suggest that the milk microbiota exhibits diurnal fluctuation resulting in a time-of-day specific taxonomic arrangement. Acknowledgments. Work supported by Cinvestav and CONACyT 163235 INFR-2011-01 grant; and CONACYT doctorate scholarship 777953 (KCC), 291236 (FHQ), and master’s degree scholarship 997494 (NGZT).