Blood lactate and pyruvate are of critical importance for the diagnosis of mitochondrial diseases. To determine guidelines for adequate blood pyruvate and lactate determinations, intraindividual studies were carried out on 10 subjects, and the influences of venostasis, delay before deproteinization, and pH in the pyruvate assay were analyzed. Delays of 1 hour or more before deproteinization of samples induced major elevations of lactate-pyruvate ratios. The lactate-pyruvate ratio correlated positively with pH in the pyruvate assay, and inadequate pH appeared as a largely underestimated cause of misleading results, while venostasis was a minor source of errors.