Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. We compared the isometric contractile and fatigue properties of1) control CK-sufficient (Ctl),2) M-CK-deficient (M-CK[−/−]), and3) combined M-CK/ScCKmit-deficient null mutant (CK[−/−]) diaphragm (Dia) to determine the effect of the absence of M-CK activity on Dia performance in vitro. Baseline contractile properties were comparable across groups except for specific force, which was ∼16% lower in CK[−/−] Dia compared with M-CK[−/−] and Ctl Dia. During repetitive activation (40 Hz, duty cycle), force declined in all three groups. This decline was significantly greater in CK[−/−] Dia compared with Ctl and M-CK[−/−] Dia. The pattern of force decline did not differ between M-CK[−/−] and Ctl Dia. We conclude that Dia isometric muscle function is not absolutely dependent on the presence of M-CK, whereas the complete absence of CK acutely impairs isometric force generation during repetitive activation.