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Background: Exercise training increases brain-derived neurotrophic factors associated with stroke recovery. However, exercise training, as a stressful stimulus, might lead to spleen contraction, and affect peripheral immune responses to impact stroke recovery.Methods: 48 C57 male adult mice were subjected to middle cerebral artery occlusion (MCAo) for 60 mins. 3 days later, they were randomly divided into two groups: 1) placed on running wheel for 20 mins daily up to 2 weeks after MCAo (RW) 2) standard housing without running wheel (no-RW). At day 5 and 7 after MCAo, the spleen and the brain were harvested. Spleen mass, splenocytes and microglia were analyzed. In another experiment, circling and corner testing were performed as neuro-functional evaluations up to 3 weeks after MCAo.Results: At day 5, compared to no-RW group, RW mice have smaller ratio of spleen weight to body weight (2.0±0.4 vs.2.6±0.4, p=0.10) but higher CD3/CD8 T-cells (13.3%±2.3 vs. 8.3%±4.0, p=0.15), and significant higher neutrophils (CD11b+/Ly6Clow/Ly6G+ cells) (2.5%±0.2 vs.1.8%±0.2, p=0.02). In the brain, RW mice have higher CD11b+ cells (26.3%±3.7% vs. 17.6%±5.0, p=0.07). At day 7, compared to no-RW group, RW did not change the spleen mass, CD3/CD8 T-cells and neutrophils, but decreased inflammatory monocytes (CD11b+/CD11c-/Ly6Chigh/Ly6G-) (0.13%±0.06 vs. 0.24%±0.04%, p=0.09) in the spleen. In the brain, although RW did not change the total CD11b+ level, RW decreased the ratio of CD11b+/CD45low to CD11b+/CD45high cells compared to no-RW group (7.7±0.7 vs. 11±2.6, p=0.08). Furthermore, on the corner test, RW group delayed the spontaneous recovery compared to no-RW group.Conclusion: Exercise training in the early phase after stroke may affect the spleen, which may in turn to affect stroke recovery. The intensity and time window of exercise training after stroke needs to be further investigated from the perspective of exercise effects on the spleen and the peripheral immune responses.