It is by now well established that tetrodotoxin-resistant (TTX-R) afferent fibres from muscle in the rat exhibit a multisensitive profile, including nociception. TTX-R afferent fibres play an important role in motor control, via spinal and supraspinal loops, but their activation and function during muscle exercise and fatigue are still unknown. Therefore, the specific effect of isometric fatiguing muscle contraction on the responsiveness of TTX-R C-fibres has been investigated in this study. To quantify the TTX-R afferent input we recorded the cord dorsum potential (CDP), which is the result of the electrical fields set up within the spinal cord by the depolarisation of the interneurons located in the dorsal horn, activated by an incoming volley of TTX-R muscle afferents. The changes in TTX-R CDP size before, during and after fatiguing electrical stimulation of the gastrocnemius-soleus (GS) muscle have been taken as a measure of TTX-R C-unit activation. At the end of the fatiguing protocol, following an exponential drop in force, TTX-R CDP area decreased in the majority of trials (9/14) to 0.75 +/- 0.03% (mean +/- SEM) of the pre-fatigue value. Recovery to the control size of the TTX-R CDP was incomplete after 10 min. Furthermore, fatiguing trials could sensitise a fraction of the TTX-R C-fibres responding to muscle pinch. The results suggest a long-lasting activation of the TTX-R muscle afferents after fatiguing stimulation. The role of this behaviour in chronic muscle fatigue in connection with pain development is discussed. Accumulation of metabolites released into the interstitium during fatiguing stimulation might be one of the reasons underlying the C-fibres' long-lasting activation.