Using sled dogs as exercise model, our objectives of this study were to (1) assess the effects of one acute bout of high-intensity exercise on surface GLUT4 concentrations on easily accessible peripheral blood mononuclear cells (PBMC) and (2) compare our findings with published research on exercise induced GLUT4 in skeletal muscle. During the exercise bout, dogs ran 5 miles at approximately 90% of VO2 max. PMBC were collected before exercise (baseline), immediately after exercise and after 24 h recovery.GLUT4 was measured via ELISA. Acute exercise resulted in a significant increase on surface GLUT4 content on PBMC. GLUT4 was increased significantly immediately after exercise (~50%; p<0.05) and reduced slightly by 24 h post-exercise as compared to baseline (~22%; p>0.05). An effect of acute exercise on GLUT4 levels translocated to the cell membrane was observed, with GLUT4 levels not yet returned to baseline after 24 h post-exercise. In conclusion, the present investigation demonstrated that acute high-intensity exercise increased GLUT4 content at the surface of PBMC of sled dogs as it has been reported in skeletal muscle in other species. Our findings underline the potential use of peripheral blood mononuclear cell GLUT4 protein content as minimally invasive proxy to investigate relationships between insulin sensitivity, insulin resistance, GLUT4 expression and glucose metabolism.