Activation of adenosine monophosphate-activated protein kinase (AMPK) plays a central role in allowing cells to adapt to nutrient deprivation in vitro. This link between AMPK activity and nutritional status has raised the possibility that AMPK plays a role in the metabolic adaptation to acute and chronic nutritional stress. However, the effects of nutritional stress on AMPK activity in vivo have not been systematically evaluated. To address this, we measured the effects of 24 h of fasting and 4 mo of caloric restriction (CR) on AMPKα1 and -α2 activities in heart, skeletal muscle, and liver in mice. Although fasting caused the expected changes in body weight, plasma leptin, and free fatty acids, it did not increase AMPK activity in heart or skeletal muscle and only increased liver AMPK activity by ∼20% (P = 0.10). Likewise, CR caused the expected changes in body weight, plasma leptin, and free fatty acids but did not alter AMPK activity in any of the three tissues. Although CR did not alter liver AMPK activity, it dramatically decreased the amount of phosphorylated acetyl-CoA carboxylase, and this was found to be due to decreased protein expression. Plasma leptin, a putative activator of AMPK, varied eightfold across the four groups of mice in the absence of changes in AMPK activity in any tissue. We conclude that, although the metabolic adaptations to fasting and CR include changes in plasma leptin concentration and phosphorylated acetyl-CoA carboxylase, these effects occur without changes in AMPK activity.