A treadmill with vibration isolation and stabilization designed for the International Space Station (ISS) was evaluated during Shuttle mission STS-81. Three crew members ran and walked on the device, which floats freely in zero gravity. For the majority of the more than 2 hours of locomotion studied, the treadmill showed peak to peak Linear and angular displacements of less than 2.5 cm and 2.5°, respectively. Vibration transmitted to the vehicle was within the microgravity allocation limits that are defined for the ISS. Refinements to the treadmill and harness system are discussed. This approach to treadmill design offers the possibility of generating 1G-like loads on the lower extremities while preserving the microgravity environment of the ISS for structural safety and vibration free experimental conditions.