The Na⁺-K⁺-ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na⁺-K⁺-ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 × 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na⁺-K⁺-ATPase maximal activity (3- O-MFPase), content ([³H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3- O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased α₁, α₂, and α₃ mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged β-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased ( P < 0.05) 3- O-MFPase activity by 5.5% (SD 2.9), and α₃ and β₃ mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na⁺-K⁺-ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3- O-MFPase activity and increase in α₁ and α₃ mRNA each persisted ( P < 0.05); the postexercise 3- O-MFPase activity was also higher after HIT ( P < 0.05). Thus HIT augmented Na⁺-K⁺-ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na⁺-K⁺-ATPase activity postexercise may contribute to reduced fatigue after training. The Na⁺-K⁺-ATPase mRNA response to interval exercise of increased α- but not β-mRNA was largely preserved posttrain, suggesting a functional role of α mRNA upregulation.