Multisport events continue to grow in popularity among recreational, amateur, and professional athletes around the world. This study aimed to determine the compounding effects of the initial run and cycling legs of an International Triathlon Union (ITU) Duathlon simulation on maximal oxygen uptake (VO_2max), ventilatory threshold (VT) and running economy (RE) within a thermoneutral, laboratory controlled setting. Seven highly trained multisport athletes completed three trials; Trial-1 consisted of a speed only VO_2max treadmill protocol (SOVO_2max) to determine VO_2max, VT, and RE during a single-bout run; Trial-2 consisted of a 10 km run at 98% of VT followed by an incremental VO_2max test on the cycle ergometer; Trial-3 consisted of a 10 km run and 30 km cycling bout at 98% of VT followed by a speed only treadmill test to determine the compounding effects of the initial legs of a duathlon on VO_2max, VT, and RE. A repeated measures ANOVA was performed to determine differences between variables across trials. No difference in VO_2max, VT (%VO_2max), maximal HR, or maximal RPE was observed across trials. Oxygen consumption at VT was significantly lower during Trial-3 compared to Trial-1 (p = 0.01). This decrease was coupled with a significant reduction in running speed at VT (p = 0.015). A significant interaction between trial and running speed indicate that RE was significantly altered during Trial-3 compared to Trial-1 (p < 0.001). The first two legs of a laboratory based duathlon simulation negatively impact VT and RE. Our findings may provide a useful method to evaluate multisport athletes since a single-bout incremental treadmill test fails to reveal important alterations in physiological thresholds.