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The mixed meal tolerance test is the gold standard measure of endogenous insulin secretion. Practical issues limit the routine clinical use of this test, including omitting insulin prior to the ingestion of a high-carbohydrate liquid mixed meal, which can result in marked hyperglycaemia. We aimed to assess whether insulin omission is necessary during the mixed meal tolerance test and whether fasting C-peptide was a practical alternative to the test.Ninety-one adults with insulin-treated diabetes (Type 1 n = 56, Type 2 n = 35) underwent two mixed meal tolerance tests; one standard without insulin and one with the patient's usual morning insulin.The 90-min serum C-peptide was highly correlated in the standard mixed meal tolerance test and the test with insulin (r = 0.98, P0.0001). There was a 20% reduction in the peak C-peptide value when insulin was given {test with insulin [0.39 (0.01-1.16) vs. test without insulin 0.48 (0.01-1.36) nmol/l, P = 0.001]}, but the original serum C-peptide cut-off for significant endogenous insulin secretion (≥ 0.2 nmol/l) still correctly classified 90/91 patients (98% sensitivity/100% specificity). Fasting serum C-peptide was highly correlated to 90-min serum C-peptide during the test (r = 0.97, P0.0001). A fasting serum C-peptide ≥ 0.07 nmol/l was the optimal cut-off (100% sensitivity and 97% specificity) for significant endogenous insulin secretion (defined as 90-min stimulated serum C-peptide ≥ 0.2 nmol/l).Insulin omission may not always be necessary during a mixed meal tolerance test and fasting serum C-peptide may offer a practical alternative in insulin-treated patients.