Skeletal muscle and exercise performance adapt to high intensity interval training (HIIT). Downhill running is an eccentric-biased exercise modality whereas uphill running is concentric-biased and flat running has aspects of both. Therefore, variation in adaptation may differ with HIIT done on flat (F) or uphill (UH) or downhill (DH) gradients. PURPOSE: To compare the training effect of three modes of HIIT on laboratory and outdoor performance and muscle cross-sectional area (CSA) and satellite cell number per fiber (SC). METHODS: 17 fit, but not elite, young adult male runners volunteered for HIIT: 6 sets of 3 minutes and 1-minute rest between, for 10 sessions over 4 weeks. Gradients were flat, +5% or -10% and running speeds 85%, 80% or 90% of peak treadmill speed (PTS) respectively. Performance tests included PTS (0% gradient), maximum isometric quadriceps force (Iso) and 5 km road time trial (TT). Muscle biopsies were taken at baseline and 6 hours after the 10^th session. Data analyzed using mixed models ANOVA, presented as mean ±SD. RESULTS: HIIT improved PTS (km/h) in F (pre: 17.7 ±1.3, post 18.9 ±1.5; P<0.05) and UH (pre: 17.6 ±0.8, post: 18.7 ±0.8 P<0.01), but not DH (pre: 17.8 ±1.3, post 18.0 ±1.3), whereas only DH significantly increased Iso strength (25% P<0.05 compared to F: -1% and UH: -4.4%). Similarly, only DH increased muscle fiber cross-sectional area (CSA) (31% P<0.05 compared to F: -5% and UH: 10%). DH increased muscle SC number/fiber highly significantly (pre: 0.097 ±0.01 post: 0.297 ±0.04 P<0.0001). Interestingly, UH HIIT group did not change SC content (pre: 0.102 ±0.018, post: 0.106 ±0.02), but flat HIIT increased significantly (pre: 0.115 ±0.01, post: 0.148 ±0.01 P<0.01), although not nearly as much as DH HIIT (F: 30%, DH: 208%). 5 km TT improved in all groups (F: 3%; UH: 3.5% and DH: 3.5%; all P<0.05). CONCLUSION: Muscle adapted differently in response to the different gradients of HIIT. Neither SC number/fiber nor CSA changed with UH HIIT. CSA also did not change with F HIIT, although SC number/fiber increased. DH HIIT increased both CSA and SC number/fiber to a greater extent. Although laboratory performance test changes differed between groups, all 3 groups improved outdoor TT performance. Therefore, different training specific adaptations in skeletal muscle conferred similar race performance improvements.