Unstable shoes (US) continually perturb gait which can train the lower limb musculature, but muscle co-contraction and potential joint stiffness strategies are not well understood. A shoe with a randomly perturbing midsole (IM) may enhance these adaptations. This study compares ankle and knee joint stiffness, and ankle muscle co-contraction during walking and running in US, IM and a control shoe in 18 healthy females. Ground reaction forces, three-dimensional kinematics and electromyography of the gastrocnemius medialis and tibialis anterior were recorded. Stiffness was calculated during loading and propulsion, derived from the sagittal joint angle-moment curves. Ankle co-contraction was analysed during pre-activation and stiffness phases. Ankle stiffness reduced and knee stiffness increased during loading in IM and US whilst walking (ankle, knee: p=0.008, 0.005) and running (p<0.001; p=0.002). During propulsion, the opposite joint stiffness re-organisation was found in IM whilst walking (both joints p<0.001). Ankle co-contraction increased in IM during pre-activation (walking: p=0.001; running: p<0.001), and loading whilst walking (p=0.003), not relating to ankle stiffness. Results identified relative levels of joint stiffness change in unstable shoes, providing new evidence of how stability is maintained at the joint level.