(1)H NMR and HPLC-MS were used to generate metabolite fingerprints for the metabonomic analysis of urine obtained from both male and female Zucker obese (fa/fa) rats, used as a model of type II diabetes, and normal male Wistar-derived animals. The resulting data were subjected to chemometric analysis (principal components analysis and partial least squares discriminant analysis) to investigate the effects of strain, diurnal variation is strain, diurnal variation and gender and gender on metabolite profiles. In the case of strain, (1)H NMR spectroscopic analysis revealed increased taurine, hippurate and formate and decreased betaine, alpha-ketoglutarate, succinate and acetate in samples from Zucker-obese compared to Wistar-derived rats. HPLC-MS analysis detected increased hippurate and ions at m/z 255.0640 and 285.0770 in positive, and 245.0122 and 261.0065 in negative electrospray ionisation (ESI), respectively, for the Zucker obese samples. Both techniques enable the detection of diurnal variation in the urine of male and female Zucker rats, marked by increases in taurine, creatinine, allantoin and alpha-ketoglutarate by (1)H NMR, and ions at m/z 285.0753, 291.0536 and 297.1492 (positive ESI) and 461.1939 (negative ESI) using HPLC-MS, in the evening samples. Differences between male and female Zucker rats were also observed. Compared to samples from male rats hippurate, succinate, alpha-ketoglutarate and dimethylglycine ((1)H NMR) were elevated in the urine of female animals together with ions at, e.g., m/z 431.1047, 325.0655, 271.0635 and 447.0946 (positive ESI) and m/z 815.5495 and 459.0985 (negative ESI) by HPLC-MS. Both analytical techniques used in this study were able to detect differences between normal and Zucker obese rats, which may provide markers of metabolic disease.