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Reduced feed intake is a hallmark following weaning, enteric diseases such as e. coli, and numerous environmental conditions such as heat stress, and has been shown to cause intestinal barrier dysfunction. Currently, several different markers and assays are utilized to evaluate intestinal barrier function, and these methods have not been thoroughly validated. Thuseed restriction may present a potential model to validate and compare multiple in vivo, ex vivo, and tissue markers of intestinal integrity. Forty-eight barrows (9.7 kg initial body weight) were fed for 7 d at 100%, 75%, 50% or 25% of expected ad libitum feed intake. Colon tissues were harvested for examination of intestinal function including ex vivo tissue transepithelial electrical resistance (TEER), tissue fluorescein isothiocyanate-dextran (FD4) transport, colonic morphology, and gene expression. Data were analyzed as an ANOVA in addition to pre-planned contrasts in which 25 and 50% feed intakes were combined in comparison to 75 and 100% feed intakes. There were no statistical differences between treatments in TEER or FD4 transport due to level of feed intake. However, there was a significant decrease in crypt depth in the colon of pigs fed at 25 and 50% compared to 75 and 100% (11%; P < 0.05). Gene expression of CLDN4 tended to decrease at 25 and 75% feeding levels. Gene expression of TLR2 was significantly increased at 25 and 50% compared to 75 and 100% feeding levels (P < 0.05) and lysozyme (LYZ) tended to be increased at 25 and 50% compared to 75 and 100% (P < 0.10). Twenty-one other genes associated with nutrient transport, immune function, and epithelial barrier function were not found to be different between feeding groups. Overall, there were minimal changes in several markers of intestinal function in the colon of restricted fed nursery pigs.