The primary aim of the present study was to test the hypothesis that training with reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for training-induced heat shock protein (HSP) adaptations of skeletal muscle. A secondary aim was to investigate the influence of reduced carbohydrate availability on oxidative adaptations and exercise performance. Three groups of recreationally active men performed 6 wk of high-intensity intermittent running occurring four times per week. Group 1 (n = 8; Low + Glu) and 2 (n = 7; Low + Pla) trained twice per day, 2 days/wk, and consumed a 6.4% glucose or placebo solution, respectively, immediately before every second training session and at regular intervals throughout exercise. Group 3 (n = 8; Norm) trained once per day, 4 days/wk, and consumed no beverage throughout training. Training induced significant improvements in maximal oxygen uptake (V̇o_2max) (P = 0.001) and distance covered on Yo-Yo Intermittent Recovery Test 2 (P = 0.001) in all groups, with no difference between conditions. Similarly, training resulted in significant increases in HSP70, HSP60, and αB-crystallin in the gastrocnemius (P = 0.03, 0.02, and 0.01, respectively) and vastus lateralis (P = 0.01, 0.02, and 0.003, respectively) muscles in all groups, with no difference between conditions. In contrast, training resulted in significant increases in succinate dehydrogenase (SDH) activity of the gastrocnemeius (Low + Glu, Low + Pla, and Norm: 27, 76, and 53% increases, respectively; P = 0.001) and vastus lateralis muscles (Low + Glu, Low + Pla, and Norm: 17, 70, and 19% increases, respectively; P = 0.001) where the magnitude of increase in SDH activity was significantly larger for both muscles (P = 0.03 and 0.04 for gastrocnemius and vastus lateralis, respectively) for subjects training in the Low + Pla condition. Data provide the first evidence that in whole body exercise conditions, carbohydrate availability appears to have no modulating effect on training-induced increases of the HSP content of skeletal muscle. In contrast, training under conditions of reduced carbohydrate availability from both endogenous and exogenous sources provides an enhanced stimulus for inducing oxidative enzyme adaptations of skeletal muscle although this does not translate to improved performance during high-intensity exercise.