Running is performed on different natural surfaces (outdoor) and artificial surfaces (indoor). Different surface characteristics cause modification of the lower leg muscle activation pattern to adopt ankle stiffness to these characteristics. So the purpose of our investigation was to study changes of lower leg muscles activation pattern in running on different natural running surfaces. Six male and two female runners participated. The participants ran at a freely chosen velocity in trials on asphalt while in trials on gravel, and grass surfaces they were attempting to reach similar velocities as in the trials on asphalt. Muscle activation of the peroneus brevis, tibialis anterior, soleus, and gastrocnemius medialis of the right leg was recorded. Running on asphalt increased average EMG amplitude of the m. tibialis anterior in the pre-activation phase and the m. gastrocnemius medialis in the entire contact phase compared to running on grass from 0.222 ± 0.113 V to 0.276 ± 0.136 V and from 0.214 ± 0.084 V to 0.238 ± 0.088 V, respectively. The average EMG of m. peroneus brevis in pre-activation phase increased from 0.156 ± 0.026 V to 0.184 ± 0.455 V in running on grass in comparison to running on gravel. Running on different surfaces is connected with different activation patterns of lower leg muscles. Running on asphalt requires stiff ankle joints, running on gravel requires greater stability in ankle joints, while running on grass is the least demanding on lower leg muscles.