AbstractCirculating trimethylamine N-Oxide (TMAO) levels predict cardiovascular disease (CVD), possibly by impacting on cholesterol metabolism and oxidative stress. Because hepatic TMAO production is regulated by insulin signalling and it is unclear whether and to what extent circulating TMAO levels associate with CVD risk, independently of insulin resistance and its important determinants fatty liver and visceral obesity, we have now addressed this question in 220 subjects who participated in the Tübingen Lifestyle Intervention Program. Visceral fat mass (r = 0.40, p < 0.0001), liver fat content (r = 0.23, p = 0.0005) and TMAO levels (r = 0.26, p < 0.0001) associated positively and insulin sensitivity associated negatively (r = −0.18, p = 0.009) with carotid intima-media thickness (cIMT). Higher TMAO levels (std.−Beta 0.11, p = 0.03) predicted increased cIMT, independently of age, sex and visceral fat mass. While during the lifestyle intervention most cardiovascular risk parameters improved, mean TMAO levels did not change (p = 0.18). However, cIMT decreased significantly (p = 0.0056) only in subjects in the tertile with the largest decrease of TMAO levels (>20%). We provide novel information that increased serum TMAO levels associate with increased cIMT, independently of established cardiovascular risk markers, including insulin resistance, visceral obesity and fatty liver. Furthermore, the decrease of cIMT during a lifestyle intervention may be related to the decrease of TMAO levels.