Several gastrointestinal symptoms associated with prolonged intense exercise(IE) have been reported, although the mechanisms underlying its effects on the intestine remain poorly understood. The aim of this study was to investigate whether IE may induce oxidative stress on the intestine, as well as its possible relationship with intestinal signaling impairments, leading to contractile disturbances. C57BL/6 mice were submitted to 4(EX.4D) and 10 days (EX.10D) of IE. The daily exercise session consisted of a running session until exhaustion with the treadmill speed set at 85% of each animal maximum velocity. The decrease in exhaustion time was exponential, and the reduction in the maximum velocity, as assessed by the incremental test, was higher in the EX.4D than in the EX.10D animals. The ileum mucosa layer was partially destroyed only after 4 days of IE, where 37% and 11% muscle layer atrophy was observed in EX.4D and EX.10D animals, respectively. Ileum contractility was significantly impaired in the EX.4D animal group, with reduced efficacy for carbachol, bradykinin (BK)- and KCl- signaling, associated with a decrease in lipid peroxidation, and no alteration of protein oxidation. Intestinal myocytes from EX.10Danimals displayed areas containing structurally disorganized mitochondria, associated with increased levels of protein oxidation, without alteration of contractility, excepting for a reduction in the potency of BK-signaling. Finally, no clear relationship between ileum contractility and oxidative stress could be evidenced. Taken together, these results argue in favor of significant functional, biochemical, and morphological disturbances caused by exercise, thus demonstrating that intestinal tissue is very sensitive to exercise.