The ability to adapt to unexpected changes in environments is associated with the risk of running-related injuries. Although gait retraining programs can mitigate injury risk, there is a scarcity of studies focusing on neuromechanical adaptations during running with unpredictable perturbations. Hence, the current experiment aimed to analyse spatial–temporal and muscle activity adaptation during a perturbed running protocol. 23 participants performed a 5-minute unperturbed and an 8-minute perturbed running trial with a baseline velocity of 2.5 m/s. During the perturbation protocol, 30-one-sided decelerative perturbations were randomly applied to both legs. Spatial-temporal data and muscle activity of twelve lower extremity and trunk muscles were recorded during unperturbed and perturbed steps. Linear mixed models with repeated measures were applied to identify adaptation at any time point in the data. Statistical analysis indicated adaptation to the perturbation trial in comparison to baseline trial for step duration, length, width and upper- and lower-leg muscles. Adaptations characterized by decreased step duration and length and increased step width and muscle activity. This study has demonstrated participants’ ability to adapt their movement and muscle activity patterns while running with unpredictable perturbations. Therefore, introducing more diverse or novel perturbation stimuli to the human system may be necessary to continually challenge adaptation.