The duration and intensity of exercise are significant factors in oxidative, morphological, and functional changes of the gastrointestinal tract. This study aimed to investigate the effects of both exhaustive swimming and probiotic VSL#3 on rats that had been previously trained with moderate swimming. The rats were divided into four groups labeled: control (C), probiotic (P), exercise (E), and probiotic-exercise (PE). Groups P and PE were fed with probiotic mixture VSL#3. Groups E and PE had a 5-week moderate swimming program (1 h/day for 5 days/week), followed by a 1-week exhaustive swimming program (trained like in moderate program but 3 times with 150 min resting sessions, for 5 days/week). At the end of the program, the rats were euthanized. Malondialdehyde, superoxide dismutase, catalase, and reduced glutathione levels were measured in tissue samples from the gastrocnemius muscle, heart, liver, kidney, and colon. In vitro contractile activity and histomorphology of the colon were also determined. Exercise and/or probiotic decreased the oxidative stress and also increased the level of one or more of the antioxidant enzymes in some of the organs. Probiotics had more pronounced effects on colon morphology than exercise but unexpectedly this effect was non-trophic. In the colon, the thickness of the tunica muscularis and the number of goblet cells were not affected; however, probiotic administration decreased the crypt depth and tunica mucosa thickness. Exercise increased the Emax value of acetylcholine (ACh), while decreased its sensitivity. These findings suggest that exhaustive swimming does not cause oxidative stress and that probiotic consumption improves oxidative balance in trained rats. The probiotic intake does not alter the effect of exercise on the contractile activity of the colon. Colon mucosal changes induced by probiotics are independent of exercise.