PURPOSE: To quantify changes in skeletal-muscle oxygenation and pulmonary O2 uptake (VO2) after an extreme ultratrail running bout. METHODS: Before (PRE) and after (POST) the race (330-km, 24000 D+/-), profiles of vastus lateralis muscle oxygenation (ie, oxyhemoglobin [O2Hb], deoxyhemoglobin [HHb], and tissue oxygenation index [TOI]) and VO2 were determined in 14 athletes (EXP) and 12 control adults (CON) during two 4-min constant-load cycling bouts at power outputs of 1 (p1) and 1.5 (p1.5) W/kg performed in randomized order. RESULTS: At POST, normalized [HHb] values increased (p1, +38.0%; p1.5, +27.9%; P < .05), while normalized [O2Hb] (p1, -20.4%; p1.5, -14.4%; P < .05) and TOI (p1, -17.0%; p1.5, -17.7%; P < .05) decreased in EXP. VO2 values were similar (P > 0.05). An "overshoot" in normalized [HHb]:VO2 was observed, although the increase was significant only during p1.5 (+58.7%, P = .003). No difference in the aforementioned variables was noted in CON (P > .05). CONCLUSIONS: The concentric and, particularly, the eccentric loads characterizing this extreme ultratrail-running bout may have led to variations in muscle structure and function, increasing the local muscle deoxygenation profile and the imbalance between O2 delivery to working muscles and muscle O2 consumption. This highlights the importance of incorporating graded training, particularly downhill bouts, to reduce the negative influence of concentric and severe eccentric loads to the microcirculatory function and to enhance the ability of runners to sustain such loading.