The objective of this study was to investigate factors influencing fat loss during tube feeding of breast milk to preterm infants. An experimental study with 81 feeding simulations was performed, with nine continuous infusions in each of six modalities: Horizontal Higher, Horizontal Matched, Horizontal Lower, Tilted Higher, Tilted Matched, and Tilted Lower, and for comparison, 27 bolus feedings: nine flushed with air, nine with water, and nine that were not flushed, done at matched height. Each simulation utilized 16 mL of breast milk given over four hours. Continuous infusions were given with a flow rate of 4 mL/h. Bolus was given as 8 mL over the course of 15–20 min every other hour. Analysis for fat, true protein, carbohydrate, total solids, and energy was performed before and after each simulation. The percent of macronutrient loss was compared between all simulations. Continuous infusion resulted in an average fat loss of 40%. Bolus feedings resulted in an average fat loss of 11% (p ≤ 0.001). Considerable fat loss is seen during continuous tube feeding. Neither height in relation to the infant nor tilting of the pump reduce fat loss. To limit fat loss, the bolus feeding method should be utilized.