There is a need for a tool to assess dietary intake related to the habitual dietary glycaemic index (GI) and fibre in groups with large numbers of individuals. Novel metabolite-profiling techniques may be a useful approach when applied to human urine. In a long-term, controlled dietary intervention study, metabolomics were applied to assess dietary patterns. A targeted approach was used to evaluate the effects on urinary C-peptide excretion caused by the dietary treatments. Seventy-seven overweight subjects followed an 8-week low-calorie diet (LCD) and were then randomly assigned to a high-GI or low-GI diet for 6 month during which they completed 24-h urine collections at baseline (prior to the 8-week LCD) and after randomisation to the dietary intervention, at month 1, 3 and 6, respectively. Metabolite profiling in 24-h urine was performed by (1)H NMR and chemometrics. Partial least squares (PLS) analysis indicated that urinary formate could discriminate between high-GI and low-GI diets (correlation coefficient r = 0.82), and this finding was confirmed statistically (P = 0.01). PLS analysis also indicated that urinary hippurate could be associated with fibre intake, but this finding was not confirmed statistically. No associations between GI and urinary C-peptide were found. Our results emphasise that application of metabolomics is useful in the assessment of dietary exposure related to dietary GI and fibre seen at group level in a nutritional metabolomic study of human urine. As our design allowed for large variations in individually selected food items, biomarkers identified at group level may be interpreted as more general and robust markers, largely not confounded with markers from single dietary factors.