Sleep restriction is associated with impaired glucose metabolism and insulin resistance, however the underlying mechanisms leading to this impairment are unknown. This study aimed to assess whether the decrease in insulin sensitivity observed after sleep restriction is accompanied by changes in skeletal muscle PKB activity. 10 healthy young males participated in this randomized crossover study which included two conditions separated by a 3-week washout period. Participants underwent two nights of habitual sleep (CON), and two nights of sleep which was restricted to 50% of habitual sleep duration (SR) in the home environment. Whole body glucose tolerance and insulin sensitivity were assessed by an oral glucose tolerance test after the second night of each condition. Skeletal muscle tissue samples were obtained from the vastus lateralis to determine PKB activity. Findings displayed no effect of trial on plasma glucose concentrations (P = 0.222). Plasma insulin area under the curve was higher after sleep restriction compared to the control (P = 0.013). Matsuda Index was 18.6% lower in the sleep restriction (P = 0.010). Fold change in PKB activity from baseline tended to be lower in the sleep restriction condition at 30 min (P = 0.098) and 120 min (P = 0.087. In conclusion, we demonstrated decreased whole body insulin sensitivity in healthy young males following two nights of sleep restriction. Skeletal muscle insulin signaling findings are inconclusive and require further study to examine any potential changes.