We aimed to analyse the mechanical properties of skinned single muscle fibres derived from the vastus lateralis (VL) muscle in relation to those of the whole intact thigh muscle and to compare any difference between young and older adults. Sixteen young men (29.25±4.65 years), 11 older men (71.45±2.94 years), 11 young women (29.64±4.88 years), and 7 older women (67.29±1.70 years) were recruited. In vivo analyses were performed for mechanical properties such as isokinetic performance, isometric torque, and power. Specific force and maximum shortening velocity (Vo) were measured with single muscle fibres. Sex difference showed greater impact on the functional properties of both the whole muscle (p<0.01) and single muscle fibres than aging (p<0.05). Sex difference, rather than aging, yielded more remarkable differences in gross mechanical properties in the single muscle fibre study in which significant differences between young men and young women were found only in the cross-sectional area and Vo (p<0.05). Age and sex differences reflect the mechanical properties of both single muscle fibres and whole thigh muscle, with the whole muscle yielding more prominent functional properties.