Purpose: The purpose of this study was twofold: (i) determine if well-trained athletes can achieve similar peak oxygen uptake (V˙O2peak) in downhill running (DR) versus level running (LR) or uphill running (UR) and (ii) investigate if lower limb extensor muscle strength is related to the velocity at V˙O2peak (vV˙O2peak) in DR, LR, and UR. Methods: Eight athletes (V˙O2max = 68 ± 2 mL·min−1·kg−1) completed maximal incremental tests in LR, DR (−15% slope), and UR (+15% slope) on a treadmill (+1, +1.5, and +0.5 km·h−1 every 2 min, respectively) while cardiorespiratory responses and spatiotemporal running parameters were continuously measured. They were also tested for maximal voluntary isometric strength of hip and knee extensors and plantar flexors. Results: Oxygen uptake at maximal effort was approximately 16% to 18% lower in DR versus LR and UR (~57 ± 2 mL·min−1·kg−1, 68 ± 2 mL·min−1·kg−1, and 70 ± 3 mL·min−1·kg−1, respectively) despite much greater vV˙O2peak (22.7 ± 0.6 km·h−1 vs 18.7 ± 0.5 km·h−1 and 9.3 ± 0.3 km·h−1, respectively). At vV˙O2peak, longer stride length and shorter contact time occurred in DR versus LR and UR (+12%, +119%, −38%, and −61%, respectively). Contrary to knee extensor and plantar flexor, hip extensor isometric strength correlated to vV˙O2peak in DR, LR, and UR (r = −0.86 to −0.96, P < 0.05). At similar V˙O2, higher heart rate and ventilation emerged in DR versus LR and UR, associated with a more superficial ventilation pattern. Conclusions: This study demonstrates that well-trained endurance athletes, accustomed to DR, achieved lower V˙O2peak despite higher vV˙O2peak during DR versus LR or UR maximal incremental tests. The specific heart rate and ventilation responses in DR might originate from altered running gait and increased lower-limb musculotendinous mechanical loading, furthering our understanding of the particular physiology of DR, ultimately contributing to optimize trail race running performance.