Background: Despite evidence for use of foot orthoses in the treatment of anterior knee pain, there is a paucity of research into their mechanisms of action. This study (i) determined the immediate lower limb kinematics and muscle activity adaptations, and (ii) evaluated the effect of individual's comfort and foot mobility. Methods: Forty individuals diagnosed with anterior knee pain were measured for lower limb kinematics and electromyographic activity (via surface electrodes) while they jogged in three prefabricated contoured orthoses (hard, medium and soft) and a soft-flat orthosis. Subjects ranked orthoses in order of comfort. Findings: Soft orthoses were more comfortable. No immediate adaptations in kinematics and electromyographic activity were observed when orthoses were added to shoes. There were few effects of perceived comfort and foot mobility, one being a significant interaction in frontal plane hip motion (Pillai's V = 0.089, P = 0.031) with the least comfortable orthosis producing the greatest relative adduction in those with mobile feet (0.54°(standard deviation 0.87)). Other main effects were a significant increase in vastus lateralis activity when wearing the least comfortable orthosis (6.94%, P = 0.007) and a delay in offset of medial gastrocnemius in individuals with less mobile feet (1.51%, P = 0.045). Interpretation: It is becoming apparent that it is important to use more comfortable foot orthoses in a condition like anterior knee pain, where there is an associated increased hip adduction and vastus lateralis activity with least comfortable orthoses. Future research is needed to determine adaptations after ongoing wearing of orthoses.