The Na+-K+-ATPase (NKA) plays a key role in muscle excitability, but little is known in human skeletal muscle about fiber-type-specific differences in NKA isoform expression or adaptability. A vastus lateralis muscle biopsy was taken in 17 healthy young adults to contrast NKA isoform protein relative abundance between type I and IIa fibers. We further investigated muscle fiber-type-specific NKA adaptability in eight of these adults following 4-wk repeated-sprint exercise (RSE) training, comprising three sets of 5 x 4-s sprints, 3 days/wk. Single fibers were separated, and myosin heavy chain (I and IIa) and NKA (alpha(1-3) and beta(1-3)) isoform abundance were determined via Western blotting. All six NKA isoforms were expressed in both type I and IIa fibers. No differences between fiber types were found for alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, or beta(3)-isoform abundances. The NKA beta(2)-isoform was 27% more abundant in type IIa than type I fibers (P < 0.05), with no other fiber-type-specific trends evident. RSE training increased beta(1) in type IIa fibers (pretraining 0.70 +/- 0.25, posttraining 0.84 +/- 0.24 arbitrary units, 42%, P < 0.05). No training effects were found for other NKA isoforms. Thus human skeletal muscle expresses all six NKA isoforms and not in a fiber-type-specific manner; this points to their different functional roles in skeletal muscle cells. Detection of elevated NKA beta(1) after RSE training demonstrates the sensitivity of the single-fiber Western blotting technique for fiber-type-specific intervention effects.