Longitudinal profiling of urinary steroids was investigated by using a gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method. The carbon isotope ratio of three urinary testosterone (T) metabolites: androsterone, etiocholanolone, 5 beta-androstane-3 alpha,17 beta-diol (5 beta-androstanediol) together with 16(5 alpha)-androsten-3 alpha-ol (androstenol) and 5 beta-pregnane-3 alpha,20 alpha-diol (5 beta-preananediol) were measured in urine samples collected from three top-level athletes over 2 years. Throughout the study, the subjects were living in Switzerland and were residing every year for a month or two in an African country. C-13-enrichment larger than 2.5 parts per thousand was observed for one subject after a 2-month stay in Africa. Our findings reveal that C-13-enrichment caused by a diet change might be reduced if the stay in Africa was shorter or if the urine sample was not collected within the days after return to Switzerland. The steroids of interest in each sample did not show significant isotopic fractionation that could lead to false positive results in anti-doping testing. In contrast to the results obtained with the carbon isotopic ratio, profiling of urinary testosterone/epitestosterone (TIE) ratios was found to be unaffected by a diet change.