BACKGROUND: Recent recommendations favoring nonfasting lipid assessment may affect low-density lipoprotein cholesterol (LDL-C) estimation. The novel method of LDL-C estimation (LDL-C N ) uses a flexible approach to derive patientspecific ratios of triglycerides to very low-density lipoprotein cholesterol. This adaptability may confer an accuracy advantage in nonfasting patients over the fixed approach of the classic Friedewald method (LDL-C F ). METHODS: We used a US cross-sectional sample of 1 545 634 patients (959 153 fasting ≥10-12 hours; 586 481 nonfasting) from the second harvest of the Very Large Database of Lipids study to assess for the first time the impact of fasting status on novel LDL-C accuracy. Rapid ultracentrifugation was used to directly measure LDL-C content (LDL-C D ). Accuracy was defined as the percentage of LDLC D falling within an estimated LDL-C (LDL-C N or LDL-C F ) category by clinical cut points. For low estimated LDL-C (<70 mg/dL), we evaluated accuracy by triglyceride levels. The magnitude of absolute and percent differences between LDL-CD and estimated LDL-C (LDL-C N or LDL-C F ) was stratified by LDL-C and triglyceride categories. RESULTS: In both fasting and nonfasting samples, accuracy was higher with the novel method across all clinical LDL-C categories (range, 87%-94%) compared with the Friedewald estimation (range, 71%-93%; P≤0.001). With LDL-C <70 mg/dL, nonfasting LDL-C N accuracy (92%) was superior to LDL-C F accuracy (71%; P<0.001). In this LDL-C range, 19% of fasting and 30% of nonfasting patients had differences ≥10 mg/dL between LDL-CF and LDL-C D , whereas only 2% and 3% of patients, respectively, had similar differences with novel estimation. Accuracy of LDL-C <70 mg/dL further decreased as triglycerides increased, particularly for Friedewald estimation (range, 37%-96%) versus the novel method (range, 82%-94%). With triglycerides of 200 to 399 mg/dL in nonfasting patients, LDLC N <70 mg/dL accuracy (82%) was superior to LDL-C F (37%; P<0.001). In this triglyceride range, 73% of fasting and 81% of nonfasting patients had ≥10 mg/dL differences between LDL-C F and LDL-C D compared with 25% and 20% of patients, respectively, with LDL-C N . CONCLUSIONS: Novel adaptable LDL-C estimation performs better in nonfasting samples than the fixed Friedewald estimation, with a particular accuracy advantage in settings of low LDL-C and high triglycerides. In addition to stimulating further study, these results may have immediate relevance for guideline committees, laboratory leadership, clinicians, and patients.