We examined the influence of prior time-trials performed at different altitudes on subsequent exercise performance in moderate hypoxia and associated cardio-metabolic and neuromuscular responses. During a preliminary visit, 10 physically active subjects cycled to exhaustion at constant workload (80% of the power output associated with their maximal oxygen uptake at a simulated altitude of 2000 m: 245±42 W) for a reference time (Tlim = 947±336 s) at 2000 m (FiO2=16.3%). Thereafter, two separate (5-7 days) sessions were conducted, in a randomized order: a cycle time trial for Tlim duration (TT1), followed 22 min later (rest) by a 6-min cycle time trial (TT2) with TT1 either performed at 2000 m or 3500 m (FiO2=13.5%), while TT2 always performed at 2000 m. Expired gases together with vastus lateralis (VL) oxygenation (near-infrared spectroscopy) and electromyographic root mean square (RMS) activity for the VL and rectus femoris (RF) muscles were continuously measured. Knee extensors electromyographic and force responses to femoral nerve stimulation were assessed before and ∼2 min after each exercise bout. During TT1, mean power output (247±42 vs. 227±37 W; P<0.001) and several physiological responses (pulse oxygen saturation: 91.0±3.0 vs. 80.2±3.1%; oxygen uptake: 44.0±7.3 vs. 39.9±5.8 ml.min-1.kg-1) were higher at 2000 m versus 3500 m. Despite this, VL and RF RMS activity together with VL oxygenation did not differ between conditions. During TT2, mean power output (256±42 vs. 252±36 W) and accompanying cardiopulmonary, quadriceps muscle activation and VL oxygenation responses did not differ after completing TT1 at 2000 m or 3500 m. Maximal isometric voluntary contraction torque (both conditions averaged: -7.9±8.4%; P<0.01), voluntary activation (-4.1±3.1%; P<0.05) and indices of muscle contractility (peak twitch torque: -39.1±11.9%; doublet torques at 10 Hz and 100 Hz: -38.7±10.2% and -15.4±8.9%; 10/100 Hz ratio: -25.8±7.7%; all P<0.001) were equally reduced from pre- to post-TT1, whereas VL and RF M-wave characteristics did not differ. Irrespectively of the altitude of TT1, neuromuscular function remained similarly depressed at pre-TT2 and post-TT2 compared to Post-TT1. In summary, exercise capacity is impaired during a time trial conducted at 3500 m versus 2000 m, whereas neural drive to quadriceps and VL oxygenation did not differ. Neuromuscular adjustments resulting from the completion of this initial exercise bout were of similar nature with, in particular, profound alterations in muscle contractility, presumably due to excitation-contraction coupling failure (low-frequency fatigue). After 22 min of rest, there was no influence of the altitude of a prior time-trial on performance and associated cardio-metabolic responses, with also no additional muscle fatigue development, during a subsequent 6-min time trial performed in moderate hypoxia.