Rodents are frequently used to model damage and diseases of the central nervous system (CNS) that lead to functional deficits. Impaired locomotor function is currently evaluated by using scoring systems or biomechanical measures. These methods often suffer from limitations such as subjectivity, nonlinearity and low sensitivity, or focus on a few very restricted aspects of movement. Thus, full quantitative profiles of motor deficits after CNS damage are lacking. Here we report the detailed characterization of locomotor impairments after applying common forms of CNS damage in rodents. We obtained many objective and quantitative readouts from rats with either spinal cord injuries or strokes and from transgenic mice (Epha4−/−) during skilled walking, overground walking, wading and swimming, resulting in model-specific locomotor profiles. Our testing and analysis method enables comprehensive assessment of locomotor function in rodents and has broad application in various fields of life science research.