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Few studies have focused on how nutrition affects the bioavailability and investment of protein during the metamorphosis of tephritids. Our study allowed us to observe how the type and particle size of the bulking agent affected the protein composition in the hemolymph of the larva and adult of Anastrepha obliqua. Results indicated that, true protein bioavailability and protein profile was greatly modified by the bulking agent and its particle size. The physical structure of the food matrix affected the content of crude fiber (F), crude protein (P), F/P ratio, non-protein nitrogen, ammoniacal nitrogen, and α-amylase and trypsin inhibitors. Results from SDS-PAGE revealed 45 fractions with well-defined bands ranging from ∼28 to ∼401 kDa in larvae and adults, we found the main differences between the samples from different food matrices within the 75–100 kDa range. Hemolymph of adults from the coarse coconut fiber food matrix treatment showed a single band with a molecular weight close to 250 kDa, probably associated with a storage protein such as lipophorins. The food matrix with a coarse bulking agent had a high concentration of ammoniacal nitrogen, suggesting high microbial activity. In conclusion, the particle size of the bulking agent of the food matrix changes the bioavailability of protein in hemolymph in the adult regardless of the total concentration of protein. Also, when the particle size of the bulking agent favored the F/P ratio, higher larval density resulted in higher individual larval weight, larval yield, and adult emergence. Inglés