We determined the effects of exercise on pancreatic endocrine responses to metabolic stimuli in type 2 diabetic (T2D) subjects and examined the influence of the diabetic status. Fourteen subjects underwent a hyperglycaemic clamp with GLP-1 infusion and arginine injection, the morning after a one-hour walk or no exercise. Subjects were stratified by high and low quantiles of fasting plasma glucose (FPG) and HbA1c as well as current use/non-use of anti-diabetic medication. In the entire cohort, exercise did not alter insulin secretion, while glucagon levels were increased in all clamp phases (P < 0.05-P < 0.01). In low FPG subjects, exercise increased GLP-1-stimulated insulin secretion (P < 0.05) with the same trend for arginine (P = 0.08). The same trends were seen for low HbA1c subjects. Furthermore, exercise increased GLP-1- and arginine-stimulated insulin secretion (P < 0.05) in anti-diabetic drug naïve subjects. Exercise-induced increases in insulin secretion are blunted in T2D subjects with high hyperglycaemia and in those using anti-diabetic drugs. Clinical Trials Registration Number: NCT01812590.