This retrospective study examined running economy (RE) as measured by oxygen uptake (VO2) for 7 highly trained male multi-sport athletes during a laboratory duathlon simulation (running + cycling). Methods: Trial 1 = speed-only single-bout run VO2max test; Trial 3 =10 km run + 30 km cycling at 95-98% of ventilatory threshold followed by a speed-only single-bout run VO2max test. RE was represented by VO2 obtained at 5, 7, and 10 mph during the VO2max tests during Trials 1 and 3. Results: A significant interaction effect (p = 0.047) occurred for Trial * Intensity (3 x 2 repeated measures ANOVA; Factor 1 = exercise intensities [speeds 5, 7, 10 mph]; Factor 2 = VO2max for Trials 1 and 3). Post hoc paired samples t-tests approached significance for 7 and 10 mph (p = 0.054; p = 0.064, respectively). Conclusion: RE is affected in a laboratory duathlon simulation during moderate intensity running.