It is not clear how the parameters of the power-duration relationship [critical power (CP) and W′] are influenced by the performance of prolonged endurance exercise. We used severe-intensity prediction trials (conventional protocol) and the 3-min all-out test (3MT) to measure CP and W′ following 2 h of heavy-intensity cycling exercise and took muscle biopsies to investigate possible relationships to changes in muscle glycogen concentration ([glycogen]). Fourteen participants completed a rested 3MT to establish end-test power (Control-EP) and work done above EP (Control-WEP). Subsequently, on separate days, immediately following 2 h of heavy-intensity exercise, participants completed a 3MT to establish Fatigued-EP and Fatigued-WEP and three severe-intensity prediction trials to the limit of tolerance (T_lim) to establish Fatigued-CP and Fatigued-W′. A muscle biopsy was collected immediately before and after one of the 2-h exercise bouts. Fatigued-CP (256 ± 41 W) and Fatigued-EP (256 ± 52 W), and Fatigued-Wʹ (15.3 ± 5.0 kJ) and Fatigued-WEP (14.6 ± 5.3 kJ), were not different ( P > 0.05) but were ~11% and ~20% lower than Control-EP (287 ± 46 W) and Control-WEP (18.7 ± 4.7 kJ), respectively ( P < 0.05). The change in muscle [glycogen] was not significantly correlated with the changes in either EP ( r = 0.19) or WEP ( r = 0.07). The power-duration relationship is adversely impacted by prolonged endurance exercise. The 3MT provides valid estimates of CP and W′ following 2 h of heavy-intensity exercise, but the changes in these parameters are not primarily determined by changes in muscle [glycogen].