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Significant elevations of traditional biomarkers of liver injury can occur as a result of running an ultramarathon.Traditional serum biomarker levels of liver injury will significantly increase as the result of participating in this 161-km race at altitude.Prospective cross-sectional study.Level 3.A total of 64 (before) and 83 (after) volunteer runners participated in a prospective observational field-based study at the Leadville 100 ultramarathon race. Changes in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), and bilirubin levels were measured.Of 669 athletes who started the race, 352 successfully completed the race within the 30-hour cutoff (53%). Of 36 runners who had pre- and postrace blood samples taken, the mean ALT, AST, and bilirubin levels were increased from 23 ± 10 U/L, 23 ± 5 U/L, and 0.60 ± 0.29 mg/dL to 117 ± 106 U/L, 485 ± 500 U/L, and 1.60 ± 0.61 mg/dL, respectively (all P < 0.001). There was no change in the mean ALP level (P = 0.11). There were no significant correlations between postrace ALT, AST, ALP, lactate dehydrogenase (LDH), and bilirubin levels and athletes’ age, sex, body mass index, or finishing time. Significant positive linear correlations between AST, ALT, and LDH with CK were seen. Athletes in this study did not seek medical attention after the race based on an electronic survey (92% response rate).Significant elevations of traditional biomarkers of liver injury occurred as a result of running an ultramarathon at altitude. These correlated with CK, a marker of muscle injury.When reviewing laboratory studies of traditional biomarkers of liver injury in athletes after an ultramarathon, significant elevations may be seen from baseline but are likely to be of no clinical consequence.