To investigate recruitment of slow-twitch (ST) and fast-twitch (FT) muscle fibres, as well as the involvement of the various quadriceps femoris muscle portions during repeated, intense, one-legged knee-extensor exercise, 12 healthy male subjects performed two 3-min exercise bouts at approximately 110% maximum thigh O2 consumption (EX1 and EX2) separated by 6 min rest. Single-fibre metabolites were determined in successive muscle biopsies obtained from the vastus lateralis muscle (n = 6) and intra-muscular temperatures were continuously measured at six quadriceps muscle sites (n = 6). Creatine phosphate (CP) had decreased (P < 0.05) by 27, 73 and 88% in ST fibres and 25, 71 and 89% in FT fibres after 15 and 180 s of EX1 and after 180 s of EX2, respectively. CP was below resting mean-1 SD in 15, 46, 84 and 100% of the ST fibres and 9, 48, 85 and 100% of the FT fibres at rest, after 15 and 180 s of EX1 and after 180 s of EX2, respectively. A significant muscle temperature increase (deltaTm) occurred within 2-4 s at all quadriceps muscle sites. DeltaTm varied less than 10% between sites during EX1, but was 23% higher (P < 0.05) in the vastus lateralis than in the rectus femoris muscle during EX2. DeltaTm in the vastus lateralis was 101 and 109% of the mean quadriceps value during EX1 and EX2, respectively. We conclude that both fibre types and all quadriceps muscle portions are recruited at the onset of intense knee-extensor exercise, that essentially all quadriceps muscle fibres are activated during repeated intense exercise and that metabolic measurements in the vastus lateralis muscle provide a good indication of the whole-quadriceps muscle metabolism during repeated, intense, one-legged knee-extensor exercise.