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Background: Completing a marathon is one of the most challenging sports activities, yet the source of running fatigue during this event is not completely understood. The aim of this investigation was to determine the cause(s) of running fatigue during a marathon in warm weather., Methodology/principal Findings: We recruited 40 amateur runners (34 men and 6 women) for the study. Before the race, body core temperature, body mass, leg muscle power output during a countermovement jump, and blood samples were obtained. During the marathon (27 °C; 27% relative humidity) running fatigue was measured as the pace reduction from the first 5-km to the end of the race. Within 3 min after the marathon, the same pre-exercise variables were obtained., Results: Marathoners reduced their running pace from 3.5 ± 0.4 m/s after 5-km to 2.9 ± 0.6 m/s at the end of the race (P<0.05), although the running fatigue experienced by the marathoners was uneven. Marathoners with greater running fatigue (> 15% pace reduction) had elevated post-race myoglobin (1318 ± 1411 v 623 ± 391 µg L(-1); P<0.05), lactate dehydrogenase (687 ± 151 v 583 ± 117 U L(-1); P<0.05), and creatine kinase (564 ± 469 v 363 ± 158 U L(-1); P = 0.07) in comparison with marathoners that preserved their running pace reasonably well throughout the race. However, they did not differ in their body mass change (-3.1 ± 1.0 v -3.0 ± 1.0%; P = 0.60) or post-race body temperature (38.7 ± 0.7 v 38.9 ± 0.9 °C; P = 0.35)., Conclusions/significance: Running pace decline during a marathon was positively related with muscle breakdown blood markers. To elucidate if muscle damage during a marathon is related to mechanistic or metabolic factors requires further investigation.