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The purpose of this study was to compare leg, knee and ankle stiffness over the course of a prolonged treadmill run (PTR) in neutral and stability footwear. Fourteen male habitual, rearfoot runners completed two biomechanical testing sessions where they ran for 21 minutes at their preferred running speed in a neutral shoe, then changed either into the same neutral shoe or a stability shoe and ran a further 21 minutes on a force instrumented treadmill. No differences were observed in leg stiffness (p > 0.05). Knee stiffness increased during the first 21 minutes (p = 0.003), while ankle stiffness reduced at minute 21 (p = 0.004) and minute 44 (p = 0.006). No differences were observed between footwear conditions (p > 0.05). These results suggest that over the course of a PTR, shock attenuation strategies change, and this change may be detrimental to the knee joint.