We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% V̇o2 peak, n = 8), 20 min (80% V̇o2 peak, n = 11), 2 min (110% of peak work rate, n = 9), or 30 s (maximal sprint, n = 10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P = 0.06) and further at 90 min of exercise (120%, P < 0.05). α2β2γ3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas α2β2γ1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. α2β2γ1-AMPK activity and AS160 phosphorylation correlated positively ( r2 = 0.55). In exercise trials 2, 3, and 4, α2β2γ3-AMPK activity but neither AS160 phosphorylation nor α2β2γ1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that α2β2γ1- but not α2β2γ3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.