Na(+), K(+)-ATPase (NKA) isoforms (α1,α2,α3,β1,β2,β3) are involved in the maintenance of membrane potential and hence are important regulators of cellular homeostasis. Given the age-related decline in skeletal muscle function, we investigated whether the natural physiological process of aging is associated with altered abundance of NKA isoforms (α1,α2,α3,β1,β2,β3) or of the commonly used control protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, measurements were made in both whole muscle or specific fiber types obtained from skeletal muscle biopsies. Seventeen healthy older (AGED, 69.4 ± 3.5 years, mean ± SD) and 14 younger (YOUNG, 25.5 ± 2.8 years) adults underwent a muscle biopsy for biochemical analyses. Comparing homogenates from AGED and YOUNG individuals revealed higher β3 isoform (p<0.05) and lower GAPDH (p<0.05). Analysis of individual fibers in muscle from YOUNG individuals, showed greater α3 and β2 isoforms, and more GAPDH in Type II compared with Type I fibers (p<0.05). In the AGED, GAPDH was higher in Type II compared with Type I fibers (p<0.05), there were no fiber type differences in the NKA isoforms (p>0.05). Compared with the same fiber type in YOUNG, α1 was greater (Type I) and α3 lower (Type II), while in both fiber types, β2 was lower, β3 greater and GAPDH lower, in muscle from AGED individuals (all p<0.05). Overall, we demonstrate that (i) GAPDH is an inappropriate choice of protein for normalization in all skeletal muscle research and (ii) full understanding of the role of NKA isoforms in human skeletal muscle requires consideration of age and muscle fiber type.