The energy cost of running (E run), a key determinant of distance running performance, is influenced by several factors. Although it is important to express E run as energy cost, no study has used this approach to compare similarly trained men and women. Furthermore, the relationship between Achilles tendon (AT) stiffness and E run has not been compared between men and women. Therefore, our purpose was to determine if sex-specific differences in E run and/or AT stiffness existed. E run (kcal kg(-1) km(-1)) was determined by indirect calorimetry at 75%, 85%, and 95% of the speed at lactate threshold (sLT) on 11 man (mean ± SEM, 35 ± 1 years, 177 ± 1 cm, 78 ± 1 kg, [Formula: see text]1 = 56 ± 1 mL kg(-1) min(-1)) and 18 woman (33 ± 1 years, 165 ± 1 cm, 58 ± 1 kg, [Formula: see text]2 = 50 ± 0.3 mL kg(-1) min(-1)) runners. AT stiffness was measured using ultrasound with dynamometry. Man E run was 1.01 ± 0.06, 1.04 ± 0.07, and 1.07 ± 0.07 kcal kg(-1) km(-1). Woman E run was 1.05 ± 0.10, 1.07 ± 0.09, and 1.09 ± 0.10 kcal kg(-1) km(-1). There was no significant sex effect for E run or RER, but both increased with speed (P < 0.01) expressed relative to sLT. High-range AT stiffness was 191 ± 5.1 N mm(-1) for men and 125 ± 5.5 N mm(-1), for women (P < 0.001). The relationship between low-range AT stiffness and E run was significant at all measured speeds for women (r (2) = 0.198, P < 0.05), but not for the men. These results indicate that when E run is measured at the same relative intensity, there are no sex-specific differences in E run or substrate use. Furthermore, differences in E run cannot be explained solely by differences in AT stiffness.