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Measuring lactate during moderate exercise is a useful tool in the diagnosis of mitochondrial disorders. It was the aim of this study, to develop reference limits for lactate at rest, during exercise and after the exercise. We investigated 18 healthy subjects and 6 patients with a mitochondrial disorder. In controls, serum lactate levels were comparable to already reported findings. In 4 patients lactate levels were markedly increased during the exercise. Measurement of serum lactate is a simple and useful step in the diagnosis of mitochondrial disorders.