The present study aimed to characterize the changes in macromolecular composition and structure in ileal tissue induced by postoperative prolonged starvation (PS), human breast milk feeding (HM) and commercial formula feeding (CF) for 48 and 72 h (h). Forty-two Wistar albino rats underwent an ileal transection and primary anastomosis and were then divided into six subgroups. Two groups of seven rats were food-deprived for 48 and 72 h with free access to water only in metabolic cages (48 h PS, 72 h PS). Then, two groups of seven rats received early enteral trophic nutrition (EEN) either using HM, and CF at 48 h post-operation (48 h HM, 48 h CF). The other two groups of seven rats received the same trophic enteral nutrition at 72 h post-operation (72 h HM, 72 h CF). An additional seven rats were fed normal rat chow (control), after which the ileal tissues were harvested and freeze-dried overnight. Then sample spectra were recorded by Fourier transform infrared (FTIR) spectroscopy. PS at 48 and 72 h resulted in an increase in the concentration of lipids and a decrease in the concentration of proteins. CF and HM trophic feeding induced a decrease in membrane fluidity and an increase in lipid order. Ileal tissues showed similar compositional and structural changes in lipids and proteins in the PS and CF groups after 48 and 72 h. A marked decrease in nucleic acid concentration was seen in CF at 48 h compared to HM. The human milk feeding groups did not induce any significant alterations and showed compositional and structural data similar to the controls. In conclusion, EEN application seems to be safer when introduced at 48 h rather than 72 h and time of this nutrition is crucial to maintain ileum structure and therefore immunity and well-being. HM-induced trophic nutrition is seen to protect the ileal tissue from significant alterations within lipid and protein compositions, whereas CF caused notable changes. HM is absolutely the best nutritional source for gut health in this animal model.