The hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry (IC) is used for estimation of insulin-stimulated substrate utilization. Calculations are based on urinary urea nitrogen excretion (UE), which is influenced by correct urine collection. The aims of our study were to improve the timing of urine collection during the clamp and to test the effect of insulin on UE in patients with type 1 diabetes (DM1; n=11) and healthy subjects (C; n=11). Urine samples were collected (a) over 24 h divided into 3-h periods and (b) before and during two-step clamp (1 and 10 mIU.kg-1.min-1; period 1 and period 2) combined with IC. The UE during the clamp was corrected for changes in urea pool size (UEc). There were no significant differences in 24-h UE between C and DM1 and no circadian variation in UE in either group. During the clamp, serum urea decreased significantly in both groups (p<0.01). Therefore, UEc was significantly lower as compared to UE not adjusted for changes in urea pool size both in C (p<0.001) and DM1 (p<0.001). While UE did not change during the clamp, UEc decreased significantly in both groups (p<0.01). UEc during the clamp was significantly higher in DM1 compared to C both in period 1 (p<0.05) and period 2 (p<0.01). The UE over 24 h and UEc during the clamp were statistically different in both C and DM1. We conclude that urine collection performed during the clamp with UE adjusted for changes in urea pool size is the most suitable technique for measuring substrate utilization during the clamp both in DM1 and C. Urine collections during the clamp cannot be replaced either by 24-h sampling (periods I-VII) or by a single 24-h urine collection. Attenuated insulin-induced decrease in UEc in DM1 implicates the impaired insulin effect on proteolysis. and Obsahuje bibliografii a bibliografické odkazy