Physical activity confers a broad spectrum of health benefits. Beyond the obvious role in metabolically driven diseases, the role of physical activity in acute liver injury is poorly explored. To study the role of physical activity in acute liver injury, a novel model of voluntary distance running in mice was developed and mice were subjected to acute liver injury induced by N-galactosamine (GalN) and lipopolysaccharide (LPS). Analyses included histological stains, immunoblotting, qRT-PCR and FACS analysis. Voluntary distance running increased to an average of 10.3 km/day after a learning curve. Running lead to a decrease in the absolute numbers of intrahepatic CD4+ T and B lymphocytes and macrophages after 7 weeks. In parallel, hepatic mRNA expression of inflammatory cytokines including IL-6 and IL-1beta, TGF-beta and monocyte chemoattractant protein-1 (MCP-1/CCL2) were suppressed, while TNF-α was not affected by exercise. Likewise, expression of the macrophage-specific antigen F4/80 was downregulated 1.6-fold from exercise. Notably, acute liver injury from GaIN/LPS was significantly blunted following 7 weeks of voluntary exercise as determined by liver histology, a 84.6% reduction of alanine aminotransferase (P<0.01) and a 54.6% reduction of aspartate aminotransferase (P<0.05) compared with sedentary mice. Additionally, proinflammatory cytokines, activation of caspase 3 and JNK were significantly lower, while antiapoptotic protein A20 increased. Voluntary distance running alters the intrahepatic immune phenotype producing an environment that is less susceptible to acute liver injury.