Context. With a long duration return mission to Mars on the horizon, we must learn as much about the environment and its influence on the musculoskeletal system as possible to develop countermeasures and mitigate deleterious health effects and maladaptation. Aims. To determine the influence of simulated Mars gravity on the activity of four locomotor muscles while walking, in comparison to 1 G, using lower body positive pressure (LBPP). Design and Methods. Fourteen males (age: 20.6 ± 2.4 years) performed bouts of walking in both simulated Mars gravity (0.38 G) and Earth gravity (1 G) using an LBPP device. Dependent variables were the muscle activity evoked in the tibialis anterior, vastus lateralis, gluteus maximus and lateral portion of the gastrocnemius, measured using electromyography and expressed as percentages of maximum voluntary isometric contractions, and heart rate (HR). For statistical analysis, a paired t-test was performed. Statistical significance was defined as P < 0.05. Results. No significant differences in muscle activity were found across conditions for any of the investigated muscles. A significant mean difference in HR was identified between Earth (105.15 ± 8.1 bpm) and Mars (98.15 ± 10.44 bpm) conditions (P = 0.027), wherein HR was lower during the Mars trial. Conclusions. The Mars environment may not result in any deteriorative implications for the musculoskeletal system. However, if future research should report that stride frequency and thus activation frequency is decreased, in simulated Mars gravity, negative implications may be posed for muscle retention and reconditioning efforts on the Red Planet.