Purpose: This study aimed to determine the contribution of metabolic, cardiopulmonary, neuromuscular, and biomechanical factors to the energy cost (ECR) of graded running in well-trained runners. Methods: Eight men who were well-trained trail runners (age: 29 [10] y, mean [SD]; maximum oxygen consumption: 68.0 [6.4] mL·min−1·kg−1) completed maximal isometric evaluations of lower limb extensor muscles and 3 randomized trials on a treadmill to determine their metabolic and cardiovascular responses and running gait kinematics during downhill (DR: −15% slope), level (0%), and uphill running (UR: 15%) performed at similar O2 uptake (approximately 60% maximum oxygen consumption). Results: Despite similar O2 demand, ECR was lower in DR versus level running versus UR (2.5 [0.2] vs 3.6 [0.2] vs 7.9 [0.5] J·kg−1·m−1, respectively; all P < .001). Energy cost of running was correlated between DR and level running conditions only (r2 = .63; P = .018). Importantly, while ECR was correlated with heart rate, cardiac output, and arteriovenous O2 difference in UR (all r2 > .50; P < .05), ECR was correlated with lower limb vertical stiffness, ground contact time, stride length, and step frequency in DR (all r2 > .58; P < .05). Lower limb isometric extension torques were not related to ECR whatever the slope. Conclusion: The determining physiological factors of ECR might be slope specific, mainly metabolic and cardiovascular in UR versus mainly neuromuscular and mechanical in DR. This possible slope specificity of ECR during incline running opens the way for the implementation of differentiated physiological evaluations and training strategies to optimize performance in well-trained trail runners.