We investigated the effects of diet (AIN93G or high-fat), physical activity (sedentary or voluntary running), and protein source (casein or soy protein isolate (SPI)) and their interactions on metabolic disturbance and inflammation in mice. After 14 weeks of feeding, the high-fat diet increased body weight gain by 34.5% (p < 0.01), whereas running reduced weight gain by 30.5% (p < 0.01) compared to their respective AIN93G and sedentary controls; SPI did not affect weight gain. The high-fat diet significantly increased plasma concentrations of insulin, glucose, triglycerides, leptin, and monocyte chemotactic protein-1 (MCP-1); running and SPI significantly reduced these parameters compared to their respective controls. The high-fat diet significantly increased and running significantly reduced plasma plasminogen activator inhibitor-1. A unique finding was that SPI supplementation to the high-fat diet reduced plasma insulin by 11% (p < 0.05), MCP-1 by 21% (p = 0.03), and tumor necrosis factor-α (TNF-α) by 50% (p = 0.05) compared to casein. As adipose tissues produce many adipocytokines, including MCP-1 and TNF-α, that contribute to a state of chronic low grade systemic inflammation and facilitate metabolic disturbance in obesity, further investigations are warranted into the roles of soy protein in reducing the risk of obesity.