To compare kinematics, kinetics and muscle activity during standing and walking for healthy subjects using an unstable test shoe (Masai Barefoot Technology, MBT) and a stable control shoe. Eight subjects volunteered for this study. During quiet standing, center of pressure excursion and muscle activity were determined. During walking, lower extremity kinematics, kinetics, and muscle electromyographic (EMG) signals were determined. Data were collected for the two shoe conditions after a 2 week accommodation period. Statistics included repeated measures ANOVAs ( α = 0.05) and post hoc tests where appropriate. During quiet standing, the center of pressure excursion was significantly and substantially greater in the unstable compared to the control shoe. Electromyographic intensity increased in the unstable test shoe for all tested muscles, but only significantly for the tibialis anterior. During locomotion, kinematics were similar in the two shoe conditions except for the initial plantar–dorsiflexion, which showed a significant more dorsiflexed position during the first half of stance in the unstable test shoe compared to the stable control shoe. The angular impulses did not show any significant differences between the two shoe conditions for all three joints but some trends towards a reduction for the knee and hip joint. There were no significant differences in electromyographic activities between the control and the unstable shoe. However, several muscles showed some trends. The unstable shoe produced changes and trends in kinematic, kinetic and electromyographic characteristics that seemed to be advantageous for the locomotor system. Further studies should investigate muscle strength, dynamic stability, pain reduction for arthritic knees and injury prevention for high performance athletes when using the unstable shoes.