The aim of the present study was to investigate the effects of altitude and distance on uphill vertical speed (VS) and the main spatio-temporal gait parameters during an extreme mountain ultra-marathon. The VS, stride height (SH) and stride frequency (SF) of 27 runners were measured with an inertial sensor at the shank for two different altitude ranges (low 1300–2000 m vs high 2400–3200 m) of 10 mountains passes distributed over a 220 km course. There was a significant interaction (F(4,52) = 4.04, p < 0.01) for the effect of altitude and distance on VS. During the first passes, the mean VS was faster at lower altitudes, but this difference disappeared at a quarter of the race length, suggesting that neuromuscular fatigue influenced the uphill velocity to a larger extent than the oxygen delivery. The average VS, SH and SF were 547 ± 135 m/h, 0.23 ± 0.05 m and 0.66 ± 0.09 Hz. The individual VS change for each uphill portions was more strongly correlated with the changes in SH (r = 0.80, P < 0.001, n = 321) than SF (r = 0.43, P < 0.001, n = 321). This suggests a large effect of the knee extensors strength loss on the diminution of VS.