Fatigue occurring during exercise can be defined as the inability to maintain the initial force or power output. In principle, force loss during fatigue can result from a decrease of the number of cross-bridges generating force or a decrease of the individual cross-bridge force or to both mechanisms. The present experiments investigated this point in single fibres or small fibre bundles isolated from flexor digitorum brevis (FDB) of C57BL/6 mice at 22-24 °C. We measured force and fibre stiffness during a fatiguing protocol (a series of 110 consecutive tetanic contractions elicited every 1.5 s) by applying small sinusoidal length oscillations at 2.5 kHz or 4 kHz frequency to the preparation and measuring the resulting force changes. The force reduction during the early phase of fatigue, was accompanied by an increased rate of tetanic force development and relaxation. In the latter stages of fatigue, the rate of force development and relaxation became slower. The analysis of the results showed that the start of fatigue is characterized by a decreased cross-bridge force, successively as fatigue becomes more marked, the number of cross-bridges decrease. These findings are discussed in the context of the current hypotheses about fatigue mechanisms.