Endurance exercise is typically associated with a shift in myosin heavy chain (MyHC) towards slower phenotype and/or an increase in mitochondrial density in skeletal muscle. Wheel running exercise has been extensively used to study these processes in mice in vivo. However, results concerning the ‘classic’ endurance‐type fast‐to‐slow fiber transition are ambiguous. We hypothesized that the specific running pattern of the mouse–i.e. short bouts at high speed‐ underlies this phenomenon and that subsequent adaptation processes involve Ca2+ handling mechanisms. Soleus muscles of male C57Bl/6 mice were isolated after six weeks of voluntary wheel running and characterized physiologically. Trained muscles had improved fatigue resistance, improved capacity for relaxation during serial electrical stimulation, and consumed 1.3‐fold more oxygen than control muscles. Biochemical analysis at the protein level revealed that neither MyHC composition nor sarcoplasmic reticular Ca2+ ATPase (SERCA) content had changed during training. Quantitative RT‐PCR revealed that phospholamban (PLB) mRNA content was 60% lower than control, while SERCA mRNA content was unchanged. This study showed that chronic wheel running in mice improves fatigue resistance of soleus muscle via an improved capacity for relaxation through downregulation of PLB expression resulting in disinhibition of SERCA.