Background: Sampling time and containers used for sample collection are crucial preanalytical processes, dictating the accuracy and precision of lipid profile results. We compared the effect of sampling time and routine container on lipid profiles. Methods: This cross-sectional study was conducted among final-year students of the Department of Biochemistry at the Federal University Otuoke, Bayelsa State, in 2024. A total of 150 participants were recruited for this study. Fasting, starvation, and random blood samples were collected from each patient into plain containers, lithium heparin, K2EDTA, and fluoride oxalate containers simultaneously on daily staged structures. Samples in anticoagulant tubes were immediately centrifuged, and plasma was obtained while samples in plain containers were made to clot before centrifugation to obtain serum. The plasma and serum obtained were used to estimate the concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-c), and high-density lipoprotein (HDL-c) using an automated chemistry analyzer. Results: The study shows a significant mean increase (P<0.05) in TC and LDL concentrations in the fasting and starvation groups compared to the control and random samples. Similarly, the anticoagulants used revealed a significant mean increase (P>0.05) in the concentrations of TC and LDL in lithium heparin and fluoride oxalate compared to EDTA and plain containers. Furthermore, the findings revealed a significant increase in triacylglycerol concentration in the lipid diet group when compared to other groups, whereas other parameters were stable. Conclusions: Random sampling in lipid profile estimation could be more suitable for fasting or starvation sampling. In a similar vein, the suitability index of sample containers placed K2EDTA and plain containers as a better choice for lipid profile estimation, comparable to lithium heparin and fluoride oxalate.