The aim of this study was to evaluate the physiological, metabolic and performance responses to duathlon performance under a range of ambient temperatures. Ten male recreational athletes performed three self-paced duathlon time trials consisting of a 5 km run (R1), a 30 km cycle and a 5 km run (R2) at 10°C, 20°C and 30°C and a relative humidity of 50%. Performance times, heart rate (HR), rating of perceived exertion (RPE), core temperature (T c) and skin temperature (T sk) were measured every kilometre. Carbohydrate and fat oxidation rates were calculated via expired gas analysis at the first and fourth kilometres during both running stages. Blood samples were taken before and after exercise for the determination of prolactin concentration. Overall performance was significantly faster at 10°C (100.76±5.32 min) than at 30°C (105.38 ± 4.28 min). Significantly higher T c was noted in the 30°C trial than in the 10°C trial, with concomitant elevations in prolactin after exercise (19.88 ± 6.48 ng/ml at 30°C; 13.10 ± 8.75 ng/ml at 10°C). The rates of carbohydrate oxidation did not differ between conditions, although fat oxidation rates were highest at 10°C. Elevated ambient temperature has a negative effect on duathlon performance. This effect may be reflected in increased T c and prolactin concentration.