Introduction The Na+-K+-ATPase (NKA) plays a key role in skeletal muscle excitability but little is known on the fibre-specific NKA abundance in human skeletal muscle. Exercise training is a proven modality for upregulating NKA in skeletal muscle and enhancing exercise performance. Repeated-sprint exercise (RSE) training comprises multiple “all-out” efforts interspersed by brief recovery periods and might differentially upregulate NKA isoforms in different fibre types. Therefore we investigated the fibre-specific differences in the abundance of all six NKA isoforms expressed in single human muscle fibres and whether fibre-specific adaptations in NKA occurred in response to intense RSE training. Methods Eight healthy participants trained three times per week for 4 weeks, each session comprising three sets of five, 4-s maximal sprints. A vastus lateralis biopsy was taken before and after training, single fibres were separated and fibre type (MHC I and IIa) and NKA isoform protein abundance (α1-3, β1-3) were determined by western blotting. Results All six NKA isoforms were co-expressed in both Type I and IIa fibres. None of the three α isoforms were expressed in a fibre type specific manner; although there was a tendency for α1 protein to be more abundant in type I compared to type IIa fibres (Figure 1). The NKA β1 and β3 isoforms were also not expressed in a fibre type specific manner (Figure 2). The NKA β2 abundance was higher in type I than in Type IIA fibres (1.33±0.18 vs. 0.83±0.07, respectively, P=0.012). RSE training upregulated NKA β1 in both type I (Pre-train 0.91±0.08, vs. Post-Train1.22±0.08, P=0.035); and Type IIa fibres (Pre-train 0.81±0.06 vs. Post-Train 1.17±0.08, P=0.05). There was a tendency for upregulation of other NKA isoforms in MHC IIa fibres post training with moderate effect sizes detected for α1 (ES=0.6), β2 (ES=0.4) and β3 (ES=0.5). Discussion The first important finding was that all six NKA isoforms were detected at the protein level in each human single muscle fibre; furthermore, these fibres showed homogeneity of NKA isoform expression across the type I and IIa fibre types. The only major exception was the greater abundance in β2 in Type I fibres. These findings contrast sharply with previous studies conducted on rat muscle. Four weeks of RSE training upregulated β1 in both fibre types, which may have implications for increasing NKA activity. However RSE training did not increase the NKA α isoform abundance in either fibre type; this may reflect insufficient physiological stress induced by 4-s sprints with RSE.