The use of motorized treadmills as convenient tools for the study of locomotion has been in vogue for many decades. However, despite the widespread presence of these devices in many scientific and clinical environments, a full consensus on their validity to faithfully substitute free overground locomotion is still missing. Specifically, little information is available on whether and how the neural control of movement is affected when humans walk and run on a treadmill as compared to overground. Here, we made use of linear and non-linear analysis tools to extract information from electromyographic recordings during walking and running overground, and on an instrumented treadmill. We extracted synergistic activation patterns from the muscles of the lower limb via non-negative matrix factorization. We then investigated how the motor modules (or time-invariant muscle weightings) were used in the two locomotion environments. Subsequently, we examined the timing of motor primitives (or time-dependent coefficients of muscle synergies) by calculating their duration, the time of main activation, and their Hurst exponent, a non-linear metric derived from fractal analysis. We found that motor modules were not influenced by the locomotion environment, while motor primitives were overall more regular in treadmill than in overground locomotion, with the main activity of the primitive for propulsion shifted earlier in time. Our results suggest that the spatial and sensory constraints imposed by the treadmill environment might have forced the central nervous system to adopt a different neural control strategy than that used for free overground locomotion, a data-driven indication that treadmills could induce perturbations to the neural control of locomotion.