Recent advances in mobile electroencephalogram (EEG) systems, featuring non-prep dry electrodes and wireless telemetry, have enabled and promoted the applications of mobile brain-computer interfaces (BCIs) in our daily life. Since the brain may behave differently while people are actively situated in ecologically-valid environments versus highly-controlled laboratory environments, it remains unclear how well the current laboratory-oriented BCI demonstrations can be translated into operational BCIs for users with naturalistic movements. Understanding inherent links between natural human behaviors and brain activities is the key to ensuring the applicability and stability of mobile BCIs. This study aims to assess the quality of steady-state visual-evoked potentials (SSVEPs), which is one of promising channels for functioning BCI systems, recorded using a mobile EEG system under challenging recording conditions, e.g., walking. To systematically explore the effects of walking locomotion on the SSVEPs, this study instructed subjects to stand or walk on a treadmill running at speeds of 1, 2, and 3 mile (s) per hour (MPH) while concurrently perceiving visual flickers (11 and 12 Hz). Empirical results of this study showed that the SSVEP amplitude tended to deteriorate when subjects switched from standing to walking. Such SSVEP suppression could be attributed to the walking locomotion, leading to distinctly deteriorated SSVEP detectability from standing (84.87 ± 13.55%) to walking (1 MPH: 83.03 ± 13.24%, 2 MPH: 79.47 ± 13.53%, and 3 MPH: 75.26 ± 17.89%). These findings not only demonstrated the applicability and limitations of SSVEPs recorded from freely behaving humans in realistic environments, but also provide useful methods and techniques for boosting the translation of the BCI technology from laboratory demonstrations to practical applications.