Aerobic exercise increases oxidant stress and leads to apoptosis of mouse intestinal lymphocytes (IL). The purpose of this study was to determine whether freewheel running prevents IL loss 24 h after a bout of strenuous exercise. Mice were randomly assigned to in-cage running wheels with 24 h access (WR) or individual cages without running wheels (NR) for 4 months. WR mice accumulated 364 km over 4 months and had higher cytochrome oxidase activity in the plantaris (p < .05), indicative of training. Total intestinal, CD3alphabeta, CD3gammadelta, CD8alpha, and CD8beta lymphocytes and intracellular glutathione were significantly lower in WR and NR mice 24 h post-exercise. The number of CD4 IL decreased 24 h after exercise in NR (p < .01) but not in WR mice relative to their respective no exercise controls. Thus, freewheel running in mice for 4 months prevented CD4 IL loss after acute exercise.