Very little attention has been given to the combined effects of healthy ageing and short-term disuse on the contractile function of human single muscle fibres. Therefore, the present study investigated the effects of 2 weeks of lower limb cast immobilisation (i.e. disuse) on selected contractile properties of single muscle fibres (n = 378) from vastus lateralis of nine young (24 ± 1 years) and eight old (67 ± 2 years) healthy men with comparable levels of physical activity. Prior to immobilisation, MHC IIa fibres produced higher maximum Ca(2+)-activated force (approx. 32%) and specific force (approx. 33%) and had lower Ca(2+) sensitivity than MHC I fibres (P <0.05), with no differences between young and old. After immobilisation, the decline in single fibre force (MHC I: young 21% and old 22%; MHC IIa: young 22% and old 30%; P <0.05) as well as specific force (MHC I: young 14% and old 13%; MHC IIa: young 18% and old 25%; P <0.05) was more pronounced in MHC IIa fibres compared to MHC I fibres (P <0.05), with no differences between young and old. Notably, there was a selective decrease in Ca(2+) sensitivity in MHC IIa fibres of young (P <0.05) and in MHC I fibres of old individuals (P <0.05), respectively. In conclusion, 2 weeks of lower limb immobilisation caused greater impairments in single muscle fibre force and specific force in MHC IIa than MHC I fibres independently of age. In contrast, immobilisation-induced changes in Ca(2+) sensitivity that were dependent on age and MHC isoform.