Stiffness of the human Achilles tendon (AT) was determined in vivo before and after a single bout of hopping exercise. It was hypothesized, based on published data using in vitro specimens, that a reduction in AT stiffness may occur after just 1000 loading cycles at physiological stress levels. Ten healthy subjects performed two-legged hopping exercise consisting of 1150-2600 high impacts. Tendon stiffness was determined in several isometric ramp contractions [20%, 40%, 60%, 80% and 100% maximum voluntary contraction (MVC)] during which tendon elongation was measured using ultrasonography and two cameras. Tendon force was calculated by dividing measured ankle torque by magnetic resonance imaging-derived AT lever arm length. Tendon stiffness remained unchanged, being 430Ჰ0 N mm-1 before and 390ᱹ0 N mm-1 after the exercise [not significant (n.s.)]. Despite the lack of changes in stiffness, maximum tendon force during MVC was reduced from 3.5ᰮ6 kN to 2.8ᰮ7 kN (P<0.01). As the proposed decline in stiffness was not observed, it is concluded that mechanical fatigue did not take place in the AT of healthy individuals after a single bout of high-impact exercise performed until exhaustion.