Objective: Accurate estimation of stiffness across anatomical levels (i.e., joint, muscle, and tendon) in vivo has long been a challenge in biomechanics. Recent advances in electromyography (EMG)-driven musculoskeletal modeling have allowed the non-invasive estimation of stiffness during dynamic joint rotations. Nevertheless, validation has been limited to the joint level due to a lack of simultaneous in vivo experimental measurements of muscle and tendon stiffness. Methods: With a focus on the triceps surae, we employed a novel perturbation-based experimental technique informed by dynamometry and ultrasonography to derive reference stiffness at the joint, muscle, and tendon levels simultaneously. Here, we propose a new EMG-driven model-based approach that does not require external joint perturbation, nor ultrasonography, to estimate multi-level stiffness. We present a novel set of closed-form equations that enables the person-specific tuning of musculoskeletal parameters dictating biological stiffness, including passive force-length relationships in modeled muscles and tendons. Results: Calibrated EMG-driven musculoskeletal models estimated the reference data with average normalized root-mean-square error ≈ 20%. Moreover, only when calibrated tendons were approximately four times more compliant than typically modeled, our approach could estimate multi-level reference stiffness. Conclusion: EMG-driven musculoskeletal models can be calibrated on a larger set of reference data to provide more realistic values for the biomechanical variables across multiple anatomical levels. Moreover, the tendon models that are typically used in musculoskeletal modeling are too stiff. Significance: Calibrated musculoskeletal models informed by experimental measurements give access to an augmented range of biomechanical variables that might not be easily measured with sensors alone.@en