The status of trail running races has exponentially grown in recent years. The present study aimed to: (a) evaluate the acute response of ultratrail racing in terms of neuromuscular function, muscle damage and hydration status; (b) analyze if responses could differ according to training levels. Twenty runners participated in the present study. The participants were divided into amateur training level (n = 10; 43.30 ± 4.52 years) or high level competitors (n = 10; 41.40 ± 6.18). Neuromuscular response (squat jump, countermovement jump and Abalakov jump), muscle damage (alanine aminotransferase, bilirubin, creatine kinase and leukocytes) and hydration status (sodium and creatinine) were evaluated before and after the Guara Somontano Ultratrail Race (108 km distance, with an accumulated slope of 5800 m). The height and power achieved by vertical jumps were lower after the race (p < 0.001). The post-race muscle damage and creatinine parameters increased in both groups (p < 0.001). The high-level group obtained lower percentages of change in squat jump and countermovement jump than the amateur-level group (p < 0.05). However, the increase in creatinine was greater for the high-level group (p < 0.05). Ultratrail racing reduces neuromuscular function and increases muscle damage. High-level runners showed less neuromuscular fatigue compared to amateur ones.