The estimation of muscle fascicle behaviour is decisive in a Hill-type model as they are related to muscle force by the force – length –velocity relationship and the tendon force – strain relationship. This study was aimed at investigating the influence of subject-specific tendon force – strain relationship and initial fascicle geometry (IFG) on the estimation of muscle forces and fascicle behaviour during isometric contractions. Ultrasonography was used to estimate the in vivo muscle fascicle behaviour and compare the muscle fascicle length and pennation angle estimated from the Hill-type model. The calibration – prediction process of the electromyography-driven model was performed using generic or subject-specific tendon definition with or without IFG as constraint. The combination of subject-specific tendon definition and IFG led to muscle fascicle behaviour closer to ultrasound data and significant lower forces of the ankle dorsiflexor and plantarflexor muscles compared to the other conditions. Thus, subject-specific ultrasound measurements improve the accuracy of Hill-type models on muscle fascicle behaviour.