Coenzyme Q10 (CoQ10) is essential for mitochondrial aerobic production of ATP via oxidative phosphorylation, but has had little study in horses. Its biologically active form is ubiquinol. We evaluated the effects of daily supplementation with ubiquinol on gluteal muscle CoQ10 concentrations and an indicator of phosphorylation status, citrate synthase (CS), in fit Thoroughbreds. Six horses received either 1 g ubiquinol daily for 3 weeks followed by 21 days without supplement, or had a 3 week unsupplemented period followed by 3 weeks of supplementation. A seventh horse received the same diet as the other horses, but no supplement, and served as a negative control. Middle gluteal muscle biopsies were obtained before feeding at day 0 (baseline), and after 10 and 21 days of each period. Muscle CoQ10 concentration was determined by HPLC with UV detection at 275 nm. CS was measured spectrophotometrically at 37 °C and related to mitochondrial CoQ10 concentration. Results (mean ± standard deviation) were analysed by 2-way RM ANOVA for effects of supplementation and time (P<0.05). Muscle baseline and non-supplemented CoQ10 concentrations prior to beginning supplementation were not different (458±156 pmol/mg protein). Values increased from 413±276 (baseline) to 977±227 pmol/mg after 10 days supplementation (P=0.03), but not thereafter (21 days: 867±194 pmol/mg; P=0.31). CS activity increased in concert with CoQ10 concentration (P=0.02; baseline: 67±18, 10 days: 155±68, 21 days: 163±78 nmol/(min.mg)). Muscle CoQ10 concentration was strongly correlated with CS activity (P=0.002; r²=0.53). Discontinued supplementation decreased muscle CoQ10 concentration and tended towards significance (P=0.06). Daily dietary supplementation with 1 g ubiquinol increased gluteal muscle [CoQ10] from day 0 to day 10, but not from days 10 to 21, possibly indicating saturation of mitochondria with ubiquinol. Associated increases in CS activity suggested aerobic metabolic capacity was enhanced with supplementation. Discontinuing supplementation decreased CoQ10 concentration.