The giant protein titin is the main determinant of passive mechanical properties in striated muscle. The distensibility of the titin filament is largely defined by the composition of the expressed isoforms, and by phosphorylation of the elastic I-band titin domains N2-B unique sequence (N2-Bus) and PEVK. Phosphorylation of the N2-Bus by PKG or PKA decreases titin stiffness, whereas phosphorylation of the PEVK domain by PKC increases it. Previous studies have suggested that intense exercise changes the passive elasticity of skeletal muscle, which was partly attributed to enhanced degradation and differential expression of titin isoforms. However, acute exercise influences the plasma concentrations of hormones, such as catecholamines and insulin, which in turn effects downstream kinase signaling. We therefore hypothesized that acute exercise may also affect the titin phosphorylation status on a short timescale. We tested our hypothesis using cell cultures of adult and embryonic rat cardiomyocytes, and isolated skeletal and heart tissue isolated from mice subjected to acute or chronic exercise regimes. Our data demonstrate that titin phosphorylation can be rapidly changed depending on the hormone status, and further suggest that altered titin phosphorylation could be important to dynamically modify myofilament stiffness in response to acute and chronic exercise.