Spaceflight represents a unique physiologic challenge to humans, altering hormonal profiles and tissue insulin sensitivity. Among these hormonal alterations, hypercortisolemia and insulin insensitivity are thought to negatively affect muscle mass and function with spaceflight. As insulin sensitivity influences the accumulation of muscle triglycerides, we examined this relationship during hypercortisolemia and inactivity. Six young healthy volunteers were confined to bed rest for 28 days. To mimic the stress response observed during spaceflight, hypercortisolemia (20-24 mg/dL) was induced and maintained by oral ingestion of hydrocortisone. On days 1 and 28 of bed rest, insulin sensitivity across the leg was assessed with a local (femoral arterial insulin infusion) 2-stage hyperinsulinemic-euglycemic clamp (stage 1, 35 microU/min per milliliter of leg; stage 2, 70 microU/min per milliliter of leg). Intramuscular lipid was measured with magnetic resonance spectroscopy. After bed rest, there was a decrease in insulin sensitivity, as assessed by glucose uptake during hyperinsulinemia (from 9.1 +/- 1.3 [mean +/- SEM] to 5.2 +/- 0.7 mg/kg of leg per minute [P = .015]). Intramuscular triglyceride increased from 0.077 +/- 0.011 to 0.136 +/- 0.018 (signal area of fat/signal area of standard, P = .009). Intramuscular lipid content correlated with the glucose uptake at day 28 (R = -0.85, P = .035). These data demonstrate that muscular inactivity and hypercortisolemia are associated with an increase in intramuscular triglyceride and skeletal muscle insulin resistance in previously healthy subjects.