The relationship between glycaemia and lipoprotein metabolism has not been completely clarified, and slight differences may be found between local authors, trials and evaluated parameters. Therefore this cross-sectional study investigated fasting cholesterol and glucose levels along with the determination of atherogenic index in a cohort of healthy individuals from the Czech Republic in relation to their fasting C-peptide levels. Data were collected between 2009 and 2018 and a total of 3189 individuals were stratified by C-peptide reference range (260-1730 pmol/l) into three groups -below (n = 111), within (n = 2952) and above (n = 126). Total, HDL, LDL cholesterol and atherogenic index were used to compare lipoprotein levels by relevant C-peptide concentrations. Participants using the supplements to affect lipid or glycaemia metabolism were excluded from this study. The evaluation of blood parameters in a fasting state included correlations between C-peptide and cholesterols, differences of variances (F-test) and the comparison of lipoprotein mean values (t-test) between the groups created by the C-peptide reference range. Mean values of total (4.9, 5.1, 5.3 mmol/l), LDL (2.6, 3.1, 3.4 mmol/l) cholesterol and atherogenic index (2.1, 2.8, 3.7) were higher with increasing C-peptide levels, whereas HDL was inversely associated with fasting C-peptide concentration. A positive and negative correlation between atherogenic index (r xy = 0.36) and HDL level (r xy = -0.36) with C-peptide values was found. Differences of HDL, LDL and atherogenic index were, in particular, recorded between the groups below and above the reference range of C-peptide (p ≤ 0.001). Considerable differences (p ≤ 0.001) were also observed for the same lipoprotein characteristics between the groups above and within the C-peptide reference. Generally, the type of cholesterol is crucial for the evaluation of specific changes concerning the C-peptide range. Lipoprotein concentrations differ in relation to C-peptide -not only below and above the physiological range, but also inside and outside of it. Conclusions: Fasting levels of cholesterol, plasma glucose, and atherogenic index were strongly associated with fasting C-peptide levels in healthy individuals. Our data suggest that fasting C-peptide could serve as a biomarker for the early detection of metabolic syndrome and/or insulin resistance prior to the manifestation of type 2 diabetes.