Our aim was to understand the information from differential two-sugar excretion (2-SE) in measuring intestinal permeability. In a crossover study in 12 healthy volunteers, we compared urinary excretion ratios of lactulose (L) to mannitol [(M) LMR] after ingestion in liquid formulation (LF) or in delayed-release, methacrylate-coated capsules (CAP). Both formulations were radiolabelled. Urine was collected every 2 h from 0 to 8 h, and from 8 to 24 h. Two hours after LF, gastric residual was 15.9 +/- 6.2% (SEM), and the percentage in colon was 49.6 +/- 7.8%; in 11/12 participants, liquid had entered colon within 2 h. Average CAP arrival time in colon was 5.16 +/- 0.46 h (mode 6 h). After LF, mannitol was extensively absorbed in the first 8 h; lactulose absorption was low throughout the 24 h. After the LF, the LMR (geometric mean, 95% CI per h) in the 0-2 h urine was [0.08 (0.05, 0.11)], which was lower than in 8-24 h urine [0.32 (0.16, 0.46); P < 0.05]. Urine LMRs at 8-24 h were similar after LF or CAP. We concluded that, after LF, sugar excretion in 0-2 h urine may reflect both SI and colon permeability. Colonic permeability is reflected by urine sugar excretion between 6 and 24 h. CAP delivery reduces mannitol excreted at 0-6 h, compared with LF. The 0-5 or 6 h 2-SE urine likely reflects both SI and colon permeability; the higher LMR in the 8-24 h urine relative to 0-2 h urine should be interpreted with caution and does not mean that colon is more permeable than SI.