In critically ill patients, ventilator-induced diaphragm muscle fibre dysfunction (VIDD) contributes to weaning problems, increasing hospitalisation time and related costs. VIDD pathophysiology remains partially unknown, especially the characterisation of the contractile dysfunction. In the present study, it was hypothesised that Ca²⁺ activation is affected during VIDD. Ca²⁺ sensitivity of contraction was therefore evaluated at the single skinned diaphragm muscle fibre level in piglets randomised into sham operation or 5-day mechanical ventilation. Ca²⁺ sensitivities of force and stiffness in fibres were significantly impaired in all mechanically ventilated piglets compared with sham-operated controls, suggesting a less efficient Ca²⁺ activation of cells, i.e. a lower relative number of strongly attached cross-bridges for each submaximal concentration of Ca ²⁺. In an attempt to test whether this negative effect of VIDD is reversible, single muscle fibres were exposed to the EMD 57033 Ca²⁺ sensitiser. EMD 57033 (30 μM) improved the Ca²⁺ sensitivity of force and stiffness in fibres from animals that were mechanically ventilated for 5 days as well as in shamoperated piglets. Thus, EMD 57033 partly restored the Ca²⁺ activation of cells, reducing VIDD. This finding offers a strong basis for evaluating the effect of Ca²⁺ sensitizers on diaphragm function in vivo.