Purpose: To better understand the carbohydrate (CHO) requirement of Australian Football (AF) match play by quantifying muscle glycogen utilization during an in-season AF match. Methods: After a 24-h CHO-loading protocol of 8 and 2 g/kg in the prematch meal, 2 elite male forward players had biopsies sampled from m. vastus lateralis before and after participation in a South Australian Football League game. Player A (87.2 kg) consumed water only during match play, whereas player B (87.6 kg) consumed 88 g CHO via CHO gels. External load was quantified using global positioning system technology. Results: Player A completed more minutes on the ground (115 vs 98 min) and covered greater total distance (12.2 vs 11.2 km) than player B, although with similar high-speed running (837 vs 1070 m) and sprinting (135 vs 138 m). Muscle glycogen decreased by 66% in player A (pre: 656 mmol/kg dry weight [dw], post: 223 mmol/kg dw) and 24% in player B (pre: 544 mmol/kg dw, post: 416 mmol/kg dw). Conclusion: Prematch CHO loading elevated muscle glycogen concentrations (ie, >500 mmol/kg dw), the magnitude of which appears sufficient to meet the metabolic demands of elite AF match play. The glycogen cost of AF match play may be greater than in soccer and rugby, and CHO feeding may also spare muscle glycogen use. Further studies using larger sample sizes are now required to quantify the interindividual variability of glycogen cost of match play (including muscle and fiber-type-specific responses), as well examining potential metabolic and ergogenic effects of CHO feeding.