Most of the increase of muscle fiber stiffness during the early phases of a tetanic contraction is due to a non-crossbridge sarcomere component whose stiffness (static stiffness) increases after stimulation with a time course very similar to the internal Ca²⁺ concentration (Bagni et al. 2002). This led us to speculate that Ca²⁺ concentration, in addition to promote crossbridge formation, could also leads to a stiffening of a sarcomere structure, identified with the titin filament, leading to the observed sarcomere stiffness increase. According to this hypothesis, static stiffness is expected to have different properties in muscles expressing different titin isoforms: we compared the static stiffness values in soleus and EDL adult mouse muscles, which express titin isoforms with long and short PEVK segment, respectively. Considering that Ca²⁺ binding to E-rich motifs in the PEVK segment increases its bending rigidity, the higher proportion of these motifs in EDL compared to soleus is expected to lead to a greater static stiffness in EDL. Our results showed that in agreement with the titin hypothesis, the static stiffness measured in single fibers at 24°C was more than five times greater in EDL than in soleus and about two times greater than previously reported on FDB muscle (Colombini et al. 2009). These results are in agreement with the idea that static stiffness depends on the increment of titin stiffness due to the interaction between Ca²⁺ and E-rich motifs in PEVK segment.