Introduction Specific muscular adaptations (1,2) potentially beneficial for endurance performance (3) were reported after high-intensity training (HIT) in hypoxia. Whether and how HIT in hypoxia would enhance intermittent glycolytic performance and repeated-sprint ability (RSA) is unclear. Our study evaluates the effects of repeated-sprint training in hypoxia or in normoxia on RSA. Methods 50 trained subjects (35 ± 7 years, 75 ± 9 kg, VO2max 52 ± 1 ml.kg.min-1) were assigned to three groups (hypoxic training H, normoxic training N, Control C). Training consisted in 8 cycling repeated-sprint sessions (3 x 5 x 10-s sprints) over 4 weeks in a normobaric hypoxic chamber (H at 3000 m and N at 485 m). PRE and POST training, subjects performed 10-s isolated sprints, RSA until exhaustion (10-s sprints, rest 20 s) 30-s Wingate and 3-min all-out test on a cycle ergometer. Muscle biopsies were taken at rest and after RSA. Results Training increased significantly (p<0.01) the number of repeated sprints in H (9.4 ± 4.8 vs. 13 ± 6.2 sprints) but not in N (9.3 ± 4.2 vs. 8.9 ± 3.5) or in C (11.0 ± 7.1 vs. 10.3 ± 6.2). 10-s sprint and Wingate performances were improved (p<0.01) similarly in H and N. Lactate and 3-min all-out performance were unchanged (Table 1 and Figure 1). mRNA gene expression of hypoxia inducible factor (HIF-1α, +55%, p<0.05), myoglobin (Mb, +16%, p=0.07) and carbonic anhydrase 3 (CA3, +35%, p<0.05) were upregulated in H only. Conversely, mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) were downregulated in H only (-40% and -23% respectively, p<0.01). Discussion Repeated-sprint training in hypoxia allowed further enhancement of repeated-sprint performance than the same training in normoxia. Systemic glycolytic (Wingate) and alactic (10-s sprint) changes being similar in H and N, RSA improvement can only be due to molecular adaptations at the muscular level induced to a greater extent by HIT in hypoxia. The upregulation of genes involved in oxygen signaling (HIF-1α), oxygen carrying (Mb) and pH regulation (CA3) and the concomitant downregulation of genes implicated in mitochondrial biogenesis (TFAM and PGC-1α) suggest a shift from aerobic to anaerobic glycolytic activity in the muscle. Our findings confirm previous results indicating molecular muscular adaptations after HIT in hypoxia (2,4,5) but show for the first time large enough adaptations for further improvement in systemic RSA performance.