Tendons are viscoelastic, which implies loading rate dependency, but loading rates of contractions are often not controlled during assessment of human tendon mechanical properties in vivo. We investigated the effects of sustained submaximal isometric plantarflexion contractions, which potentially negate loading rate dependency, on the stiffness of the human Achilles tendon in vivo using dynamometry and ultrasonography. Maximum voluntary contractions (high loading rate), ramp maximum force contractions with 3 s loading (lower loading rate) and sustained contractions (held for 3 s) at 25%, 50% and 80% of maximal tendon force were conducted. No loading rate effect on stiffness (25–80% max. tendon force) was found. However, loading rate effects were seen up to 25% of maximum tendon force, which were reduced by the sustained method. Sustained plantarflexion contractions may negate loading rate effects on tendon mechanical properties and appear suitable for assessing human Achilles tendon stiffness in vivo.