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The purpose of this study was to characterize the effects of prolonged β-adrenoceptor stimulation on O2 uptake and triglyceride/fatty acid (TG/FA) cycling during rest with and without previous exercise. Eight men performed two exercise (90 min cycling at 56 ± 3 (SD)% of maximal O2 uptake, followed by 4.5 h bed rest) and two rest-control experiments. In one rest and one exercise experiment a bolus dose (5 g) of the β-adrenoceptor agonist isoprenaline was given immediately after exercise, followed by a continuous infusion (20 ng kg–1 min–1), and at the corresponding time in the rest experiment. In the other experiments saline was given instead. The O2 uptake increased in the post-exercise period both with and without β-stimulation. The total excess post-exercise oxygen consumption (EPOC) was not different between saline (8.1 ± 1.8 (SE) L) and isoprenaline administration (10.8 ± 1.8 L, P = 0.40). Also, the total accumulated increase in O2 uptake for the 4.5 h period after isoprenaline infusion was not different between the rest (12.5 ± 2.0 L) and the exercise experiments (15.2 ± 1.7 L, P = 0.40). The rate of TG/FA cycling increased after both exercise and isoprenaline treatment, but no interaction effect was found. In conclusion, the increases observed in O2 uptake and the rate of TG/FA cycling during β-adrenoceptor stimulation were not increased by a previous exercise bout.