The interaction of middle-aging and chronic high-fat feeding on glucose transport in skeletal muscle and adipose tissue was examined. To this end, we studied the effects of 6 month treatment with a high-fat diet in 12-month old rats. Chronic high-fat feeding led to a substantial reduction in GLUT4 glucose transporter expression both in adipose tissue and in skeletal muscle, which was additive to the reduction in GLUT4 protein content detected in aged rats. In parallel, the high-fat diet led to a reduced insulin-stimulated glucose transport in the incubated soleus muscle and isolated adipocytes; insulin resistance induced by high-fat feeding was superimposed to the decreased insulin response detected in aged rats. Different mechanisms were responsible for GLUT4 repression in response to high-fat feeding or aging in skeletal muscles and adipose tissue.