The oxygen cost of transport per unit distance (CoT; mL·kg(-1)·km(-1)) shows a U-shaped curve as a function of walking speed (v), which includes a particular walking speed minimizing the CoT, so called economical speed (ES). The CoT-v relationship in running is approximately linear. These distinctive walking and running CoT-v relationships give an intersection between U-shaped and linear CoT relationships, termed the energetically optimal transition speed (EOTS). This study investigated the effects of subtracting the standing oxygen cost for calculating the CoT and its relevant effects on the ES and EOTS at the level and gradient slopes (±5%) in eleven male trained athletes. The percent effects of subtracting the standing oxygen cost (4.8 ± 0.4 mL·kg(-1)·min(-1)) on the CoT were significantly greater as the walking speed was slower, but it was not significant at faster running speeds over 9.4 km·h(-1). The percent effect was significantly dependent on the gradient (downhill > level > uphill, P < 0.001). The net ES (level 4.09 ± 0.31, uphill 4.22 ± 0.37, and downhill 4.16 ± 0.44 km·h(-1)) was approximately 20% slower than the gross ES (level 5.15 ± 0.18, uphill 5.27 ± 0.20, and downhill 5.37 ± 0.22 km·h(-1), P < 0.001). Both net and gross ES were not significantly dependent on the gradient. In contrast, the gross EOTS was slower than the net EOTS at the level (7.49 ± 0.32 vs. 7.63 ± 0.36 km·h(-1), P = 0.003) and downhill gradients (7.78 ± 0.33 vs. 8.01 ± 0.41 km·h(-1), P < 0.001), but not at the uphill gradient (7.55 ± 0.37 vs. 7.63 ± 0.51 km·h(-1), P = 0.080). Note that those percent differences were less than 2.9%. Given these results, a subtraction of the standing oxygen cost should be carefully considered depending on the purpose of each study.