Background: The retention of human milk nutrients in gastric tubes used to feed premature infants is a challenge to be overcome. Research Aims: To evaluate (1) the performance of six homogenizers (mixing processor, piston valve, ultrasonic bath, ultraturrax, stirring mixer, and ultrasound probe) for the fat retention reduction in gastric tubes; (2) the influence of the best homogenization conditions on the fatty acid and protein profiles of human milk; and (3) the cost/benefit ratio for the inclusion of homogenization as a new step in human milk processing. Methods: The influence of different levels and times of homogenization on reducing fat retention of human milk in probes was evaluated in this comparative prospective cross-sectional study. After homogenization, human milk flowed through a gavage and infusion pump apparatus used for feeding. Fat content was quantified before and after feeding. The techniques that reduced fat globule sizes and/or promoted a lower percentage of fat holding were evaluated for efficiency, variations in the fatty acid and protein profiles, and energy density and operating costs. Results: Homogenization led to a reduction in fat retention in feeding probes. The mixer processor and the ultrasound probe reduced fat retention by 99.23% (SD = 0.07) and 99.95% (SD = 0.02), respectively, and did not negatively influence fatty acid and protein profiles. The mixer processor demonstrated low energy density and low cost for human milk processing. Conclusion: Homogenization promoted reduced fat retention in the feed probe and could help maintain fat nutrients of human milk during enteral feeding.