Purpose: Previous research has shown that maximal oxygen uptake (VO2max) significantly influences performance in trail-running races up to 120 km but not beyond. Similarly, the influence of running economy on performance in ultratrail remains unclear. The aim of our study was, therefore, to determine the physiological predictors of performance in a 166-km trail-running race. Methods: Thirty-three experienced trail runners visited the laboratory 4 to 8 weeks before the race to undergo physiological testing including an incremental treadmill test and strength assessments. Correlations and regression analyses were used to determine the physiological variables related to performance. Results: Average finishing time was 37:33 (5:52) hours. Performance correlated significantly with VO2max (r = −.724, P < .001), velocity at VO2max (r = −.813, P < .001), lactate turn point expressed as percentage of VO2max (r = −.510, P = .018), cost of running (r = −.560, P = .008), and body fat percentage (r = .527, P = .012) but was not related to isometric strength. Regression analysis showed that velocity at VO2max predicted 65% of the variability in performance (P < .001), while a model combining VO2max and cost of running combined predicted 62% of the variability (P = .008). Conclusion: This is the first study to show that VO2max and velocity at VO2max are significant predictors of performance in a 166-km trail-running race. This suggests that ultratrail runners should focus on the development of these 2 qualities to optimize their race performance.