A unique group of circulating very-long-chain saturated fatty acids (VLCSFAs), including arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0), have been associated with a lower risk of type 2 diabetes, although associations with early metabolic risk phenotypes preceding type 2 diabetes have received limited study. We aimed to examine the associations of VLCSFAs with longitudinal changes in insulin sensitivity and β-cell function in a cohort at risk for type 2 diabetes. VLCSFAs in the four main serum pools (phospholipid, triacylglycerol, cholesteryl ester, and nonesterified fatty acid) were extracted from fasting baseline samples (n = 467). Generalized estimating equations were used to determine the associations between VLCSFAs and changes over 9 years in validated indices of insulin sensitivity (HOMA2-%S insulin sensitivity as percentage of normal population and ISI) and β-cell function (insulinogenic index IGI, IGI divided by HOMA-insulin resistance IGI/IR, and insulin secretion sensitivity index 2 ISSI-2). Associations of VLCSFAs with outcomes were strongest in the triacylglycerol lipid pool: 20:0 was positively associated with both insulin sensitivity and β-cell function (5.01% increase in HOMA2-%S and 4.01–6.28% increase in IGI/IR and ISSI-2 per SD increase in 20:0); 22:0 was positively associated with insulin sensitivity, with a 6.55% increase in HOMA2-%S and a 5.80% increase in ISI per SD increase in 22:0. Lastly, 24:0 was positively associated with insulin sensitivity and β-cell function (7.94–8.45% increase in HOMA2-%S and ISI, and a 4.61–6.93% increase in IGI/IR and ISSI-2 per SD increase in 24:0). Fewer significant associations were observed in the cholesteryl ester and nonesterified pools. Overall, our results indicate positive longitudinal associations of VLCSFAs with insulin sensitivity and β-cell function, especially within the triacylglycerol pool. Article Highlights