Mitochondria continuously change shape, position and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone, accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility and matrix protein diffusion. To this end we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity and the rate of matrix EYFP diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria was 2-3 times higher in patient cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI deficient patients become more glycolytic, allowing a higher mitochondrial velocity.