Background: Human breast milk is used sparingly in infants with intestinal failure due to observations from studies using syringe pumps that show loss of macronutrients with continuous feeding. Because of the potential benefits of using human milk, we sought to assess macronutrient losses using human milk as continuous tube feeds as done in the inpatient and home setting using a feeding bag and pump. Methods: Using in vitro simulations of human milk to assess macronutrient losses with continuous tube feeds, hourly samples were analyzed using the SpectraStar Near-Infrared Analyzer (Unity Scientific, Columbia, MD). Effects of agitation and positional changes of the feeding bag on macronutrient delivery were investigated in 249 total samples. Pairwise comparisons were performed using repeated-measures analysis of variance. Results: Significant fat losses were observed at all rates at hours 1–4, averaging to 73% at 5 mL/h. Caloric losses correlated strongly with fat losses. Significant gains in the fat content (+116% at hour 4 at 5 mL/h) were seen in the preinfusion aliquots (feeding bags). Horizontal positioning and continuous agitation of the feeding bag only partially limited fat losses. Fat delivery at 5 mL/h was significantly enhanced to 87% when the feeding bag was placed in an inverted position and improved further up to 98% with higher infusion rates. No carbohydrates and proteins losses were seen. Conclusions: Enabling the delivery of the human milk from the top of the feeding bag optimizes fat delivery and limits losses. Such top infusing feeding bags should be developed and could decrease healthcare costs for intestinal rehabilitation programs.