This study aimed to quantify net glycogen utilization in the vastus lateralis (VL) and gastrocnemius (G) of male (n = 11) and female (n = 10) recreationally active runners during three outdoor training sessions. After 2-d standardization of carbohydrate intakes (6 g·kg body mass per day), glycogen was assessed before and after 1) a 10-mile road run (10-mile) at lactate threshold, 2) 8 × 800-m track intervals (8 × 800 m) at velocity at V˙O2max, and 3) 3 × 10-min track intervals (3 × 10 min) at lactate turnpoint. Resting glycogen concentration was lower in the G of female compared with males (P < 0.001) runners, although no sex differences were apparent in the VL (P = 0.40). Within the G and VL of male runners, net glycogen utilization differed between training sessions where 10 miles was greater than both track sessions (all comparisons, P < 0.05). In contrast, net glycogen utilization in female runners was not different between training sessions in either muscle (all comparisons, P > 0.05). Net glycogen utilization was greater in male than in female runners in both VL (P = 0.02) and G (P = 0.07) during the 10-mile road run. With the exception of male runners during the 3 × 10-min protocol (P = 0.28), greater absolute glycogen utilization was observed in the G versus the VL muscle in both male and female runners and during all training protocols (all comparisons, P < 0.05). Data demonstrate that 1) prolonged steady-state running necessitates a greater glycogen requirement than shorter but higher-intensity track running sessions, 2) female participants display evidence of reduced resting muscle glycogen concentration and net muscle glycogen utilization when compared with male participants, and 3) net glycogen utilization is higher in the G muscle compared with the VL.