In vivo calorie restriction (CR; consuming 60% of ad libitum, AL, intake) induces elevated insulin-stimulated glucose transport (GT) in skeletal muscle. The mechanisms triggering this adaptation are unknown. The aim of this study was to determine if physiologic reductions in extracellular glucose and/or insulin, similar to those found with in vivo CR, were sufficient to elevate GT in isolated muscles. Epitrochlearis muscles dissected from rats were incubated for 24 h in media with glucose (8 mM) and insulin (80 µU/ml) similar to plasma values of AL-fed rats and compared to muscles incubated with glucose (5.5 mM) and/or insulin (20 µU/ml) similar to plasma values of CR rats. Muscles incubated with CR-levels of glucose and insulin for 24 h had a subsequently greater (P<0.005) GT with 80 µU/ml insulin and 8 mM [³H]-3-O-methylglucose, but unchanged GT without insulin. Reducing only glucose or insulin for 24 h, or both glucose and insulin for 6 h, did not induce altered GT. Increased GT after 24 h incubation with CR-levels of glucose and insulin was not attributable to increased insulin receptor tyrosine phosphorylation, Akt serine phosphorylation, or Akt substrate of 160 kDa phosphorylation. Nor did 24 h incubation with CR-levels of glucose and insulin alter the abundance of insulin receptor, insulin receptor substrate 1, GLUT1 or GLUT4 proteins. These results provide the proof-of-principle that reductions in extracellular glucose and insulin, similar to in vivo CR, are sufficient to induce an increase in insulin-stimulated glucose transport comparable to the increase found with in vivo CR.