Use of LC-MS/MS methods has improved sample preparation and increased throughput for the measurement of 40 or more organic acids in urine. In order to assess the significance of abnormalities that might be attributed to nutritional inadequacies or other metabolic disturbances, the week-to-week variation of results due to normal physiological responses needs to be established. This study determined the biological variability for 37 organic acids plus hippuric acid, D-arabinitol and 8-hydroxy-2'deoxyguanosine in overnight urine specimens from eight weekly samples submitted by 22 healthy adults. For the 40 analytes, CV b values varied from 12.3 to 74.3. Fourteen of the analytes had CV b values less than 30 and another 19 of them were less than 50. Multiple analytes displayed the property of increasing variability with concentration that may be characteristic of most intermediary metabolites. Linear regression line slopes for CV b vs. concentration were tabulated to assist the use of this information. The 40 analytes display biological variability in the range of disease risk markers such as serum lipoprotein cholesterol concentrations, cancer markers and thyroid hormones. The likelihood of a single measurement being representative of the true mean concentration varies with the analyte and the level found. Data reported here demonstrate reliability of results of urinary organic acid profiling performed under the reported analytical conditions. [N A J Med Sci. 2012;5(3): 148-156.]