Aims <p>To examine the effects of administering detemir and glargine insulin on the detection of endogenous C-peptide and insulin levels.</p> Methods <p>This study enrolled 69 patients with type 2 diabetes (T2DM) who use long-acting insulin therapy. Fasting and postprandial blood glucose (BG), C-peptide, and insulin were detected for two consecutive mornings. Fasting and postprandial C-peptide/BG and insulin/BG were then compared between groups treated with or without detemir and glargine insulin the night before. Thirty patients with type 1 diabetes (T1DM) verified the results.</p> Results <p>In patients with T2DM, compared with the results obtained from the group that did not administer insulin the night prior to testing, fasting C-peptide (CP0h), CP0h/fasting blood glucose (FBG), fasting insulin (INS0h), and INS0h /FBG were decreased with the use of detemir. Similarly, CP0h and CP0h/FBG were decreased in the group that had glargine administered the night before testing. However, there was no significant difference in INS0h and INS0h/FBG with and without the use of glargine insulin. The use of long-acting insulin had no effect on the postprandial C-peptide/PBG and postprandial insulin/PBG. In patients with T1DM, CP0h/FBG was hardly detected and had no difference between glargine and detemir groups. However, after using detemir the night before, the INS0h/FBG was hardly detected; insulin could be detected in the glargine group.</p> Conclusions <p>Exclusive dependence on C-peptide levels as a primary metric for the direct evaluation of pancreatic function under insulin therapy is insufficient. A comprehensive assessment necessitates due consideration of the insulin type employed for quantifying insulin and C-peptide concentrations.</p>