The aim of this study was to investigate the mRNA response related to mitochondrial biogenesis, metabolism, angiogenesis, and myogenesis in trained human skeletal muscle to speed endurance exercise (S), endurance exercise (E), and speed endurance followed by endurance exercise (S + E). Seventeen trained male subjects (maximum oxygen uptake (VO2-max): 57.2 ± 3.7 (mean ± SD) mL·min(-1)·kg(-1)) performed S (6 × 30 sec all-out), E (60 min ~60% VO2-max), and S + E on a cycle ergometer on separate occasions. Muscle biopsies were obtained at rest and 1, 2, and 3 h after the speed endurance exercise (S and S + E) and at rest, 0, 1, and 2 h after exercise in E In S and S + E, muscle peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α) and pyruvate dehydrogenase kinase-4 (PDK4) mRNA were higher (P < 0.05) 2 and 3 h after speed endurance exercise than at rest. Muscle PGC-1α and PDK4 mRNA levels were higher (P < 0.05) after exercise in S + E than in S and E, and higher (P < 0.05) in S than in E after exercise. In S and S + E, muscle vascular endothelial growth factor mRNA was higher (P < 0.05) 1 (S only), 2 and 3 h after speed endurance exercise than at rest. In S + E, muscle regulatory factor-4 and muscle heme oxygenase-1 mRNA were higher (P < 0.05) 1, 2, and 3 h after speed endurance exercise than at rest. In S, muscle hexokinase II mRNA was higher (P < 0.05) 2 and 3 h after speed endurance exercise than at rest and higher (P < 0.05) than in E after exercise. These findings suggest that in trained subjects, speed endurance exercise provides a stimulus for muscle mitochondrial biogenesis, substrate regulation, and angiogenesis that is not evident with endurance exercise. These responses are reinforced when speed endurance exercise is followed by endurance exercise.