Human milk oligosaccharides (HMOs) are complex glycans associated with positive infant health outcomes. The concentrations of HMOs in the milk of lactating women are associated with substantial intra- and inter-individual differences and may be influenced by maternal physiological and/or nutrition-related factors. The primary aim of this study was to explore potential influences of short-term maternal diet and current body composition on HMO profiles in mature human milk. Milk samples were collected at 3–4 months postpartum from 101 healthy Australian women using standardised procedures, and analysed for macronutrients (lactose, fat, and protein). In addition, HMO concentrations were analysed using liquid-chromatography mass-spectrometry (LC-MS). Maternal dietary data were collected using three validated 24-h dietary recalls, and the body composition of a subgroup of mothers was assessed by DEXA scans (n= 30). Most (79%) of the women were secretor-positive. Individual nutrients were not significantly correlated with HMO concentrations after correction for multiple comparisons (p> 0.05), except for dietary folate intake. DEXA scans revealed no associations between HMO profiles and maternal body composition during established lactation. The study findings suggest a lack of clear and consistent associations between maternal nutrition and HMO concentrations in mature human milk from healthy lactating women with adequate dietary intake. The prevailing influence of genetic variation in lactating mothers may overshadow any impact of maternal nutritional and/or physiological status on HMO composition in mature human milk.