SUMMARY1. Considerable debate exists as to whether the properties of diaphragm muscles can be modified by training. As the diaphragm is chronically activated during normal respiration, it is of interest to determine whether this muscle is resistant to further modification by exercise. The aim of this study was to investigate the contractile activation characteristics of single skinned muscle fibres from the diaphragm of both CONTROL and TRAINED rats.2. Male rats were subjected to a 20 week high‐intensity endurance exercise training programme that consisted of running on a motorized treadmill, 5 days/week, 90–120 min/day, 27–30 m/min, up a 20° incline. At the conclusion of training, rats were killed with an overdose of ether and costal regions of the diaphragm were removed and stored in a glycerol‐based skinning solution at—20° C.3. Single skinned (membrane‐permeabilized) diaphragm muscle fibres were attached to a sensitive force transducer and activated in Ca2+‐ and Sr2+‐buffered solutions in order to determine relative force‐pCa and force‐pSr characteristics. Fibres were allocated into discrete groups (population I, population II, intermediate, mixed) on the basis of their physiological (contractile) properties.4. Population I (slow‐twitch) fibres from the diaphragm of TRAINED rats exhibited a reduced sensitivity to Ca2+(indicating a rightward shift of the force‐pCa relationship) compared to those diaphragm fibres from CONTROL animals. An increased number of population II (fast‐twitch) fibres were sampled from TRAINED rats, however, training did not affect the activation properties of these fibres.5. The fact that training‐induced alterations in the contractile characteristics did occur in one group of fibres indicates that the intensity of training was sufficient to induce some modifications to the costal fibres of the diaphragm muscle. However, the lack of alterations to the contractile characteristics of the population II (fast‐twitch) fibres may also suggest that the overall properties of the diaphragm are relatively resistant to modification