This paper demonstrates an optical segment tracking (OST) approach for assessing the in vivo bone loading regimes in humans. The relative movement between retro-reflective marker clusters affixed to the tibia cortex by bone screws was tracked and expressed as tibia loading regimes in terms of segment deformation. Stable in vivo fixation of bone screws was tested by assessing the resonance frequency of the screw-marker structure and the relative marker position changes after hopping and jumping. Tibia deformation was recorded during squatting exercises to demonstrate the reliability of the OST approach. Results indicated that the resonance frequencies remain unchanged prior to and after all exercises. The changes of Cardan angle between marker clusters induced by the exercises were rather minor, maximally 0.06°. The reproducibility of the deformation angles during squatting remained small (0.04°/m–0.65°/m). Most importantly, all surgical and testing procedures were well tolerated. The OST method promises to bring more insights of the mechanical loading acting on bone than in the past.