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The biological response to ultra-endurance mountain race events is not yet well understood. The aim of this study was to determine the biochemical and physiological changes after performing an ultra-endurance mountain race in runners. We recruited 11 amateur runners (age: 29.7 ± 10.2 years; height: 179.7 ± 5.4 cm; body mass: 76.7 ± 10.3 kg). Muscle damage, lactate concentration, energy balance, rating of perceived exertion (RPE), heart rate (HR), heart rate variability (HRV), body composition changes, and jump performance were analyzed before, during (only lactate, HR, and HRV), and after the race. Athletes completed 54 km in 6 h, 44 min (±28 min). After the race, myoglobin and creatine kinase concentration increased from 14.9 ± 5.2 to 1419.9 ± 1292.1 μg/L and from 820.0 ± 2087.3 to 2421.1 ± 2336.2 UI/L, respectively ( p < 0.01). In addition, lactate dehydrogenase and troponin I significantly increased after the race ( p < 0.01). Leukocyte and platelet count increased by 180.6% ± 68.9% and 23.7% ± 11.2%, respectively ( p < 0.001). Moreover, after the competition, athletes presented a 3704 kcal negative energy balance; a significant increase in RPE values; a decrease in countermovement and squat jump height; and a decrease in body mass and lower limb girths. During the event, lactate concentration did not change and subjects presented a mean HR of 158.8 ± 17.7 beats/min, a significant decrement in vagal modulation, and a significant increase in sympathetic modulation. Despite the relative 'low' intensity achieved, ultra-endurance mountain race is a stressful stimulus that produces a high level of muscle damage in the athletes. These findings may help coaches to design specific training programs that may improve nutritional intake strategies and prevent muscle damage. [ABSTRACT FROM AUTHOR]