We tested the hypotheses that (i) a decrease in activation of skeletal muscles at short sarcomere lengths (SLs) is caused by an inhibition of Ca²⁺ release from the sarcoplasmic reticulum (SR), and (ii) the decrease in Ca²⁺ would be caused by an inhibition of action potential conduction from the periphery to the core of the fibres. Intact, single fibres dissected from the flexor digitorum brevis from mice were activated at different SLs, and intracellular Ca²⁺ was imaged with confocal microscopy. Force decreased at SLs shorter than 2.1µm, while Ca²⁺ concentration decreased at SLs below 1.9µm. The concentration of Ca²⁺ at short SL was lower at the core than at the peripheries of the fibre. When the external concentration of Na⁺ was decreased in the experimental media, impairing action potential conduction, Ca²⁺ gradients were observed in all SLs. When caffeine was used in the experimental media, the gradients of Ca²⁺ were abolished. We concluded that there is an inhibition of Ca²⁺ release from the sarcoplasmic reticulum (SR) at short SLs, which results from a decreased conduction of action potential from the periphery to the core of the fibres