The current investigation was designed (1) to examine the effect of a 48-week strength training on musculotendinous (MT) and musculoarticular (MA) stiffness characteristics in older men and women; and (2) to evaluate the influence of gender on stiffness behaviour in response to such training. The training was performed twice per week and mainly consisted of three series of 10 repetitions of calf-rise at 75% of the 3-repetition maximum. Two methods were used to perform stiffness measurements during plantar flexion: (1) the use of quick-release movements, allowing the calculation of MT stiffness; (2) the application of sinusoidal perturbations to the joint, allowing the calculation of MA stiffness. In each case, stiffness was linearly related to torque, leading to the calculation of a normalized stiffness index (SI) as the slope of this stiffness-torque relationship: SI_MT and SI_MA, respectively. Results showed a similar decrease in SI_MT among older men (-27%, P < 0.05) and women (-29%, P < 0.05) following training. A decrease in SI_MA was only observed among women (-11%, P < 0.05). The results suggest that (1) MT stiffness decreases following training in older individuals, counterbalancing the effect of ageing; and (2) older men and women respond differently to the same resistance-training stimulus in terms of MA stiffness. Gender-related differences in MA stiffness response may originate from passive MA elastic structures.