Glycation induced crosslinking of connective tissue collagen is thought to be involved in the pathogenesis of various disorders associated with diabetes and aging. Although the formation of the glycation related collagen crosslinks appears to be universal for all tissues, currently it is unknown whether differences exist between soft and hard tissue biomechanics in response to glucose-mediated in vitro glycation. In this study, the impact of non-enzymatic glycation was investigated on tendons and bones, using them as models for soft and hard tissue respectively. Achilles tendons from rabbits, and femur and tibia from rats, were subjected to in vitro glycation with glucose. Sixty days following glycation, the matrix integrity of the tendons and bones was evaluated and compared with the respective non-glycated tissue (n=10 in each group). The results revealed that the impact of glycation was significant on the tendon but not on the bone. Measurements of the biomechanical stability of glycated tendons indicated a significant increase in maximum load (21%), Young's modulus of elasticity (72%), energy to yield (35%) and toughness (68%) compared to the non-glycated tendons. No significant differences were found in breaking strength, bending stiffness, energy to yield and toughness between glycated and non-glycated femurs or tibias. The deformation of both soft and hard tissue was unaffected by the glycation. Measurements at ultimate tissue failure (break point) revealed that glycated tendons bore significantly higher load and energy absorption than non-glycated tendons. In contrast, the deformation of the glycated tendons at break point was considerably reduced as compared to control tendons. However, glycation had no significant effects on the hard tissue biomechanical properties at break point. The results of this study demonstrate that in vitro glycation influences the biomechanical properties of soft tissue but not hard tissue.