Na+/K+ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, β, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and β1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAβ1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K+ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min–1, p < 0.01) while also increasing cumulated leg K+ reuptake 0–3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAβ1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = –0.53, p < 0.05). NKAα2 and β1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAβ1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K+ shifts and enhance muscle performance during intense exercise.