Objective - To assess changes in muscle glycogen (MG) and triglyceride (MT) concentrations in aerobically conditioned sled dogs during prolonged exercise. Animals - 54 Alaskan sled dogs fed a high-fat diet. Procedures - 48 dogs ran 140-km distances on 4 consecutive days (cumulative distance, up to 560 km); 6 dogs remained as nonexercising control animals. Muscle biopsies were performed immediately after running 140, 420, or 560 km (6 dogs each) and subsequently after feeding and 7 hours of rest. Single muscle biopsies were performed during recovery at 28 hours in 7 dogs that completed 560 km and at 50 and 98 hours in 7 and 6 dogs that completed 510 km, respectively. Tissue samples were analyzed for MG and MT concentrations. Results - In control dogs, mean ± SD MG and MT concentrations were 375 ± 37 mmol/kg of dry weight (kgDW) and 25.9 ± 10.3 mmol/kgDW, respectively. Compared with control values, MG concentration was lower after dogs completed 140 and 420 km (137 ± 36 mmol/kgDW and 203 ± 30 mmol/kgDW, respectively); MT concentration was lower after dogs completed 140, 420, and 560 km (74 ± 5.4 mmol/kgDW; 9.6 ± 6.9 mmol/ kgDW, and 6.3 ± 4.9 mmol/kgDW, respectively). Depletion rates during the first run exceeded rates during the final run. Replenishment rates during recovery periods were not different, regardless of distance; only MG concentration at 50 hours was significantly greater than the control value. Conclusions and Clinical Relevance - Concentration of MG progressively ncreased in sled dogs undergoing prolonged exercise as a result of attenuated depletion.