Borate is an antibacterial preservative widely used in clinical and large-scale epidemiological studies involving urine sample analysis. Since it readily forms covalent adducts and reversible complexes with hydroxyl and carboxylate groups, the effects of borate preservation in (1)H NMR-spectroscopy-based metabolic profiling of human urine samples have been assessed. Effects of various concentrations of borate (range 0-30 mM) on (1)H NMR spectra of urine were observed at sequential time points over a 12 month period. Consistent with known borate chemistry, the principal alterations in the (1)H resonance metabolite patterns were observed for compounds such as mannitol, citrate, and alpha-hydroxyisobutyrate and confirmed by ESI-MS analysis. These included line-broadening, T(1) and T(2) relaxation, and chemical shift changes consistent with complex formation and chemical exchange processes. To further investigate complexation behavior in the urinary metabolite profiles, a new tool for visualization of multicomponent relaxation variations in which the spectra were color-coded according to the T(1) and T(2) proton relaxation times respectively (T(1) or T(2) ordered projection spectroscopy, TOPSY) was also developed and applied. Addition of borate caused a general decrease in (1)H T(1) values, consistent with nonspecific effects such as solution viscosity changes. Minor changes in proton T(2) relaxation rates were observed for the most strongly complexing metabolites. From a molecular phenotyping and epidemiologic viewpoint, typical interpersonal biological variation was shown to be vastly greater than any variation introduced by the borate complexation, which had a negligible effect on the metabolic mapping and classification of samples. While caution is indicated in the assignment of biomarker signals where metabolites have diol groupings or where there are adjacent hydroxyl and carboxylate functions, it is concluded that borate preservation is "fit-for-purpose" for (1)H NMR-based epidemiological studies, since the essential biochemical classification features of the samples are robustly maintained.