Tendinous structures often exhibit reduced stiffness following repeated loading via static muscular contractions. The purpose of this study was to determine if human Achilles tendon (AT) stiffness is affected by the repeated loading experienced during running and if this affects normal muscle–tendon interaction. Twelve male participants (mean ± SD: age 27 ± 5 years, height 1.79 ± 0.06 m, mass 78.6 ± 8.4 kg) completed a 30 min run at 12 kmph on a treadmill. AT properties were determined before and after the run during a series of one-legged hops. During hopping and running, AT length data were acquired from a combination of ultrasound imaging (50 Hz) and kinematic data (200 Hz). AT force was estimated from inverse dynamics during hopping and AT stiffness was computed from plots of AT force and length. AT stiffness was not significantly different post run (pre 163 ± 41 N mm−1, post 147 ± 52 N mm−1, P > 0.05) and peak AT strain during the stance phase of running (calculated relative to AT length during standing) was similar at different time points during the run (3.5 ± 1.8% at 1 min, 3.2 ± 1.8% at 15 min and 3.8 ± 2% at 30 min). It was concluded that the loading experienced during a single bout of running does not affect the stiffness of the AT and that the properties of the AT are stable during locomotion. This may have implications for muscle fascicle behaviour and Achilles tendon injury mechanisms.