Locomotion is one of the human's most important functions that serve survival, progress and interaction. Gait requires kinematic and dynamic coordination of the limbs and muscles, multi-sensory fusion and robust control mechanisms. The force stimulus generated by the interaction of the foot with the walking surface is a vital part of the human gait. Although there have been many studies trying to decipher the load feedback mechanisms of gait, there is a need for the development of a versatile system that can advance research and provide new functionality. In this paper, we present the design and characterization of a novel system, called Variable Stiffness Treadmill (VST). The device is capable of controlling load feedback stimulus by regulating the walking surface stiffness in real time. The high range of available stiffness, the resolution and accuracy of the device, as well as the ability to regulate stiffness within the stance phase of walking, are some of the unique characteristics of the VST. We present experiments with healthy subjects in order to prove the concept of our device and preliminary findings on the effect of altered stiffness on gait kinematics. The developed system constitutes a uniquely useful research tool, which can improve our understanding of gait and create new avenues of research on gait analysis and rehabilitation.