The study aimed to compare the ankle muscles activation, biomechanics and energetics of running in male runners during submaximal level run using minimalist (MinRS) and traditional cushioned (TrdRS) running shoes. During 45-min running in MinRS and TrdRS, the ankle muscles pre- and co-activation, biomechanics, and energetics of running of 16 male endurance runners (25.5 ± 3.5 yr) were assessed using surface electromyography (tibialis anterior and gastrocnemius lateralis), instrumented treadmill and indirect calorimetry, respectively. The net energy cost of running (C _r ) was similar for both conditions (P = 0.25) with a significant increase over time (P < 0.0001). Step frequency (P < 0.001), and total mechanical work (P = 0.001) were significantly higher in MinRS than in TrdRS with no evolution over time (P = 0.28 and P = 0.85, respectively). The ankle muscles pre- and co-activation during the contact phase did not differ between the two shoe conditions (P ≥ 0.33) or over time (P ≥ 0.15). In conclusion, during 45-min running, Cr and muscle pre- and co-activation were not significantly different between MinRS and TrdRS with significantly higher step frequency and total mechanical work noted in the former than in the latter. Moreover, C _r significantly increased during the 45-min trial in both shoe conditions along with no significant change over time in muscle activation and biomechanical variables.