Regulation of maximal Na⁺-K⁺-ATPase activity in vastus lateralis muscle was investigated in response to prolonged exercise with (G) and without (NG) oral glucose supplements. Fifteen untrained volunteers (14 males and 1 female) with a peak aerobic power (VO_2peak) of 44.8±1.9 ml.kg^-1.min^-1, mean±SE) cycled at ~57% VO_2peak to fatigue during both NG (artificial sweeteners) and G (6.13±0.09% glucose) in randomized order. Consumption of beverage began at 30 min and continued every 15 min until fatigue. Time to fatigue was increased (P<0.05) in G compared to NG (137±7 vs 115±6 min). Maximal Na⁺-K⁺-ATPase activity (V_max) as measured by the 3-O-methylfluorescein phosphatase assay (nmol.mg^-1.h^-1) was not different between conditions prior to exercise (85.2±3.3 or 86.0±3.9), at 30 min (91.4±4.7 vs 91.9±4.1) and at fatigue (92.8±4.3 vs 100±5.0) but was higher (P<0.05) in G at 90 min (86.7±4.2 vs 109±4.1). Na⁺-K⁺-ATPase content (_max) measured by the vanadate facilitated [³H] ouabain-binding technique (pmol.g^-1wet wt) although elevated (P<0.05) by exercise (0<30, 90 and fatigue) was not different between NG and G. At 60 and 90 min of exercise, blood glucose was higher (P<0.05) in G compared to NG. The G condition also resulted in higher (P<0.05) serum insulin at similar time points to glucose and lower (P<0.05) plasma epinephrine and norepinephrine at 90 min of exercise and at fatigue. These results suggest that G results in an increase in V_max by mechanisms that are unclear.