Blood acetate originates from fiber degradation by the hindgut microbiota. In addition to glucose and fat, acetate represents an energy substrate for muscles during exercise. Compared with horses fed high-starch diets, it has been reported that high-fiber diets lead to higher plasma acetate concentrations prior and post-exercise, lower lactatemia during exercise, and lower glycogen depletion post-exercise. Fibrolytic efficiency of the hindgut microbiota could thus play a crucial role in energy metabolism during exercise by enhancing acetate availability, which could modulate performance. This study aimed at investigating the relationships between fibrolytic efficiency, energy metabolism, and exercise performance in Standardbred horses. Twenty-one elite 2-years old Standardbred horses (12 stallions, 9 mares) from Ecurie Hunter Valley with similar feeding (hay ad libitum + 6.8kg concentrates/day) and training regimen (3 very heavy works/week) were included in this experiment. An individual in vitro fermentation test was performed from fecal samples with hay as substrate. Gas production (GP) in the bottles was monitored during 48h and fitted to a generalized model using nonlinear regression. Dry matter (DM), cellulose, and hemicellulose were analyzed in substrate residues, and their disappearance was calculated. Two weeks after, horses performed an incremental exercise including 3 steps of 3 min duration at35km/h, 40km/h, and maximal velocity. Steps were separated by 1 min walking. Maximal speed was defined as the maximal velocity sustained for 1 min during the last step. Blood samples were collected before (T0), 5 min (T5), and 20 min (T20) after the incremental exercise to measure glucose, lactate, NEFA, and acetate concentrations. Maximal speed, blood parameters, and fermentation parameters were correlated with Pearson coefficients. No significant correlation was found between maximal speed and glycemia, lactatemia, or NEFA concentration. Maximal speed was correlated with asymptotic GP (P = 0.04; r = 0.46), DM (P = 0.01; r = 0.53) and cellulose (P = 0.02; r = 0.52) disappearance, and with acetate concentration measured at T5 (P = 0.03; r = 0.48). Higher in vitro fibrolytic activity of the hindgut ecosystem was associated with better performance, although resting acetate did not correlate significantly with maximal speed. These results suggest that the extra source of energy provided by fiber digestion could benefit muscle activity during high-intensity exercise in horses. Further work should monitor blood metabolites during exercise to evaluate acetate absorption from the hindgut and utilization by muscles.