Accelerometer-based systems are often used to quantify human movement. This study's aim was to assess the reliability and validity of the Myotest® accelerometer-based system for measuring running stride kinematics. Twenty habitual runners ran two 60 m trials at 12, 15, 18 and 21 km·h(-1). Contact time, aerial time and step frequency parameters from six consecutive running steps of each trial were extracted using Myotest® data. Between-trial reproducibility of measures was determined by comparing kinematic parameters from the two runs performed at the same speed. Myotest® measures were compared against photocell-based (Optojump Next®) and high-frequency video data to establish concurrent validity. The Myotest®-derived parameters were highly reproducible between trials at all running speeds (intra-class correlation coefficient (ICC): 0.886 to 0.974). Compared to the photo-cell and high-speed video-based measures, the mean contact times from the Myotest® were 34% shorter and aerial times were 64% longer. Only step frequency was comparable between systems and demonstrated high between-system correlation (ICC ≥ 0.857). The Myotest® is a practical portable device that is reliable for measuring contact time, aerial time and step frequency during running. In terms of validity, it provides accurate step frequency measures but underestimates contact time and overestimates aerial time compared to photocell- and optical-based systems.