In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments and the reuptake of Ca²⁺ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca²⁺ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and loaded with Indo-I for imaging of sarcoplasmic Ca²⁺ changes in real-time by confocal microscopy. Myotubes were field-stimulated with 10 ms-pulses of 16V to evoke transient rises in sarcoplasmic Ca²⁺ concentration ([Ca²⁺]_S). Three single pulses, two pulse trains (1 Hz) and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca²⁺]_S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca²⁺ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity the amplitude of the [Ca²⁺]_S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased as compared to control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca²⁺ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca²⁺] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity.