Tendons are responsive to mechanical loading and their properties are often the target of intervention programs. The tendon’s mechanical properties, particularly stiffness, also govern its function, therefore changes to these properties could have substantial influence on energy-saving mechanisms during activities utilizing the stretch-shortening cycle. We investigated Achilles tendon (AT) function in vivo during walking with respect to a training intervention that elicited significant increases in AT stiffness. 14 men and women completed 12-weeks of isometric plantarflexor strength training that increased AT stiffness, measured during isometric MVC, by ~31%. Before and after the intervention, participants walked shod at their preferred velocity on a fully-instrumented treadmill. Movement kinematics, kinetics and displacement of the gastrocnemius medialis muscle-tendon junction were captured synchronously using 3D motion capture and ultrasound imaging, respectively. A MANOVA test was used to examine changes in AT force, stress, strain, stiffness, Young’s modulus, hysteresis and strain energy, measured during walking, before and following strength training. All were non-significant for a main effect of time, therefore no follow-up statistical tests were conducted. Changes in joint kinematics, tendon strain, velocity, work and power and muscle activity during the stance phase were assessed with 1D statistical parametric mapping, all of which also demonstrated a lack of change in response to the intervention. This in vivo examination of tendon function in walking provides an important foundation for investigating the functional consequences of training adaptations. We found substantial increases in AT stiffness did not impact on tendon function during walking. AT stiffness measured during walking, however, was unchanged with training, which suggests that increases in stiffness may not be evident across the whole force-elongation relation, a finding which may help explain previously mixed intervention results and guide future investigations in the functional implications of tendon adaptation.