Introduction and objective: Human milk contains all the nutrients and bioactive components required for the optimal growth and development of newborns. It is also an optimal source of nutrition in premature infants, however it does not fully meet their nutritional demands. Studies have shown that infants fed exclusively human milk have better enteral nutrition tolerance, lower risk of necrotising enterocolitis, and improved neurodevelopmental outcomes. The aim of the study was to analyse preterm content of human milk collected from women as part of a randomised controlled trial assessing tailored nutrition. Materials and methods: We studied 726 samples from 39 mothers of premature infants during their first postnatal four weeks. Infants were assigned to standard or tailored enteral nutrition, but all mothers were asked to pool milk samples from full breast expression at each feed across a 24-hour period twice a week. Glucose, fat, protein, and energy levels were measured using the Miris Human Milk Analyser and compared to published values. We assessed the relationship between postnatal week and human milk content. Results: Protein content decreased over time. Median protein at four weeks of postnatal age was 1.37 and 1.32 per 100 mL (tailored vs. standard group, respectively). Lipid content increased over time. Median lipid content at four weeks of postnatal age was 4.06 and 3.82 per 100 mL (tailored vs. standard group). Glucose remained stable. Energy content increased over time only in the tailored group. Median energy at four weeks of postnatal age was 73.35 per 100 mL. Conclusions: Variations in human milk content exist between populations and should be considered when prescribing enteral nutrition in preterm infants.