Characterisation of expression of, and consequently also the acute exercise effects on Na⁺,K⁺-ATPase isoforms in human skeletal muscle remains incomplete and were therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at ~40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 and 24 h post-exercise, and analysed for Na⁺,K⁺-ATPase & [alpha] ₁ , ₂ , & [alpha]₃ , ₁ , & [beta]₂ and ₃ mRNA and crude homogenate protein expression, using Real-Time RT- PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na⁺,K⁺-ATPase isoforms. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352±69 s; mean±SEM) immediately increased ₃ and ₂ mRNA by 2.4- and 1.7-fold, respectively (P<0.05), whilst ₁ and ₂ mRNA were increased by 2.5- and 3.5-fold at 24 h and 3 h post-exercise, respectively (P<0.05). No significant change occurred for ₁ and ₃ mRNA, reflecting variable time-dependent responses. When the average post-exercise value was contrasted to rest, mRNA increased for all ₁-₃, ₁-₃ isoforms, by 1.4-, 2.2-, 1.4-, 1.1-, 1.0- and 1.0-fold, respectively (P<0.05). However, exercise did not alter the protein abundance of the ₁ -₃ and ₁-₃ isoforms. Thus, human skeletal muscle expresses each of the Na⁺,K⁺-ATPase ₁ , ₂ , ₃ , ₁, ₂ and ₃ isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na⁺,K⁺-ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na⁺,K⁺-ATPase upregulation.