We investigated whether impaired sarcolemmal excitability causes severe fatigue during repeated tetani in isolated mouse skeletal muscle. Slow-twitch soleus or fast-twitch extensor digitorum longus (EDL) muscles underwent intensive stimulation (standard protocol: 125 Hz for 500 ms, every second, parallel plate electrodes, 20 V, 0.1-ms pulses). Interventions with altered stimulation characteristics were tested either on the entire fatigue profile or after 90- to 100-s stimulation. d-tubocurarine did not alter the fatigue profile in soleus thereby eliminating impaired neuromuscular transmission. Lower stimulation frequencies partially restored peak force, especially in soleus. The twitch force-stimulation strength relationship shifted towards higher voltages in both muscle types, with a much larger shift in EDL. Augmenting pulse strength restored tetanic force from 29% (4.4 V) to 79% (20 V), or slowed fatigue in soleus. Increasing pulse duration (0.1 to 1.0 ms) restored tetanic force from 8 to 46% in EDL and from 41 to 90% in soleus; 0.25-ms pulses restored tetanic force to 83% in soleus. Switching from transverse wire to parallel plate stimulation increased tetanic force from 34 to 63%, and fatigue was exacerbated with wires compared with plates in soleus. The combined data suggest that impaired excitability (disrupted action potential generation) within trains is the main contributor ( approximately 50% initial force) to severe fatigue in both muscle types, the surface rather than t-tubular membrane is the main site of impairment during wire stimulation, and extreme fatigue in EDL includes an increased action potential threshold leading to inexcitable fibers. Moreover, mathematical modeling discounts anoxia as the major contributor to fatigue during our stimulation regime in isolated muscles.