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Background: Infants born at very low birth weight (VLBW) are vulnerable to deficits in fatty acids (FAs) but little is known of factors that influence the intakes or composition of their human milk feeds., Objectives: We aimed to identify sources of variability in the fat composition of human milk fed to VLBW infants and examine the impact of milk source (mother's own or donor) on fat and FA intakes., Methods: Serial samples of mother's milk (n = 476) and donor milk (n = 53) fed to infants born weighing <1250 g (n = 114 infants from 100 mothers) were collected [Optimizing Mothers' Milk for Preterm Infants (OptiMoM) randomized clinical trial]. Fat and FA were analyzed using a mid-infrared human milk analyzer and GC with flame ionization detection., Results: At full enteral feeding, donor milk is estimated to provide 1.3 g · kg-1 · d-1 less total fat than mature mother's milk (recommended intake: 4.8 g · kg-1 · d-1), and 5-9 mg · kg-1 · d-1 less DHA (22:6n-3) and arachidonic acid (20:4n-6) (estimated average requirement: 55-60 and 35-45 mg · kg-1 · d-1, respectively) than colostrum or transitional milk. Similar deficits were observed in measured intakes of a subset of OptiMoM infants. In multivariable-adjusted models, maternal ethnicity had medium to large [≥0.5 SD score (SDS)] effects on DHA, SFAs, and MUFAs. Mothers with prepregnancy BMI in overweight and obese categories had higher milk total fat (β: 0.35; 95% CI: 0.10, 0.61 and β: 0.46; 95% CI: 0.16, 0.77 SDS, respectively). Those with BMI ≥30 in addition had higher proportions of SFAs (β: 0.61; 95% CI: 0.33, 0.89 SDS) and lower DHA (β: -0.54; 95% CI: -0.89, -0.20 SDS). Other factors, such as gestational age and income, were also associated with FA composition., Conclusions: The fat and FA content of human milk fed to VLBW infants is variable. Care must be taken to ensure fat and FA intakes meet recommendations, particularly when feeding a high proportion of donor milk.This trial was registered at clinicaltrials.gov as NCT02137473., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)