The relative advantages of assessing ²-cell secretory function by measuring plasma or urinary insulin or Cpeptidewas studied in five athletes during timed periods:1 period, baseline fasting (3 h); period 2, exercise (1.5 h) at 75% maximum heart rate; period 3, rest (2.5 h); period 4, eating (3 h); and period 5, rest (2 h). Blood (collected using a constant withdrawal pump) and urine samples from each period were evaluated for insulin and C-peptide immunoreactivity. Plasma insulin decreased from 0.10 ± 0.01 pmol/ml (13.6 ± 2 μU/ml) in period 1 to 0.06 ± 0.02 pmol/ml (8.7 ±2.1 μU/ml; P < 0.005) in period 2, and increased in period 4 to 0.42 ± 0.08 pmol/ml (58 ± 10.9 μU/ml). Plasma C-peptide declined from 0.55 ±0.11 pmol/ml in period 1 to 0.33 ± 0.07 pmol/ml in period 2 (P < 0.005), and rose in period 4 to 1.9 ± 0.34 pmol/ml (P < 0.005). Urinary insulin decreased from a baseline of 0.05 ± 0.02 pmol/min (7.2 ± 2.1 μU/min) to 0.03 ± 0.01 pmol/min (3.7 ± 0.9 n) in period 2 (P < 0.05), while urinary C-peptide decreased from 7.96 ± 1.15 to 3.97 ± 0.38 pmol/min (P < 0.005). Urinary insulin and C-peptide increased in period 4 to 0.16 ± 0.04 pmol/min (22.1 ± 5.1 μU/min) and 53.19 ± 12.7 pmol/min, respectively. Urinary insulin clearance remained constant during the five periods (0.46 ± 0.01 ml/min). C-Peptide clearance increased from a baseline clearance of 16.2 ± 2.6 to 22.26 ± 2.62 ml/min (P < 0.05) in period 4, but was comparable to baseline measurements in periods 2, 3, and 5 (11.8 ± 3.3, 11.3 ± 5.6, and 17.5 ± 6.6 ml/min, respectively). The mean clearance ratio of Cpeptide to creatinine (0.12 ± 0.02) was 31 times that of insulin to creatinine (0.004 ± 0.001). These data indicate that urinary Cpeptide excretion reflects integrated plasma insulin and C-peptide levels and is an excellent noninvasive measure of ²-cell secretion in various physiological circumstances. However, changes in C-peptide clearance must be taken into account when interpreting urinary values of this peptide.