We studied the intra-individual variation in plasma glucose, specific serum insulin and serum pro-insulin concentrations, measured by two 75-g oral glucose tolerance tests in an age, sex, and glucose tolerance stratified random sample from a 50–74-year-old Caucasian population without a history of diabetes mellitus. The intra-individual variation was assessed by the standard deviation of the test-retest differences (SDdif). For subjects with normal (n=246), impaired glucose tolerance (n=198), and newly detected diabetes (n=80) classified at the first test, the following (SDdif/median level of individual average scores) were found: fasting glucose: 0.4/5.4, 0.5/5.9 and 0.7/7.2 mmol/l; 2-h glucose: 1.3/5.6, 1.8/8.5 and 2.3/12.8 mmol/l; fasting insulin: 23/76, 32/89 and 30/ 116 pmol/l; 2-h insulin: 190/303, 278/553 and 304/626 pmol/l; fasting proinsulin: 4/8, 6/13 and 9/18 pmol/l; 2-h proinsulin: 19/49, 23/84 and 33/90 pmol/l, respectively. In both glucose, proinsulin and insulin concentrations the total intra-individual variation was predominantly determined by biological variation, whereas analytical variation made only a minor contribution. The SDdif can easily be interpreted, as 95% of the random test-retest differences will be less than 2 · SDdif, or in terms of percentage, less than (2 · SDdif/median level of individual average scores) · 100. Therefore, for subjects with normal glucose tolerance, 95% of the random test-retest differences will be less than 15% (fasting glucose), 46% (2-h glucose), 61% (fasting insulin), 125% (2-h insulin), 100% (fasting proinsulin) and 78% (2-h proinsulin) of the median value of the individual average scores. No substantial independent association of either age, gender or obesity with the intra-individual variation in glucose, proinsulin, or insulin concentrations was found.