Abstract. Research in human and rodents has shown an age-associated decline in physical function, aerobic capacity and skeletal muscle mitochondrial function, which in humans begins around the age of 50. On the other hand, many horses can still actively work or compete beyond 20 years of age, an age equivalent to a 65-year-old human. The purpose of the present study was to determine the age-related changes in fiber type composition and mitochondrial function in equine skeletal muscle. Muscle biopsies of right gluteus medius and triceps brachii from young (1.8 ± 0.1 y; n = 24) and aged (20 ± 5 y; n = 10) Quarter Horses were compared. High-resolution respirometry was performed on freshly sampled and subsequently permeabilized muscle fibers. Remaining tissue was frozen in liquid nitrogen and stored at −80°C for measurement of fiber type composition and enzyme activities. Statistical differences were analyzed using one-way ANOVA and Holm-Sidak post hoc analysis (Sigmaplot 12.0). We found that aged horses had a higher percentage of oxidative type I myosin heavy chain (MHC) isoform in both gluteus (P < 0.001) and triceps (P = 0.024) compared with young horses. The proportion of glycolytic type IIX MHC isoform tended to decrease with advancing age, particularly in triceps (P = 0.061). The proportion of intermediate fiber type IIA MHC isoform was not affected by age. Age had no effect on mitochondrial respiration in gluteus; but triceps from aged compared with young horses had greater leak respiration (P = 0.038), electron transport system capacity (P = 0.032), and a tendency for a lower respiratory control ratio (P = 0.076). Cytochrome c oxidase activity in both triceps (P < 0.001) and gluteus (P < 0.001) was lower in aged compared with young horses. Using citrate synthase activity as a marker, mitochondrial density increased by 26.8% in the gluteus of aged horses (P = 0.034), but was unaffected by age in the triceps (P = 0.183). Our data suggest that aging resulted in an increased percentage of oxidative type I fibers, increased mitochondrial density, and impaired mitochondrial function in Quarter Horse skeletal muscle.