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Background Aortic intima-media thickness measured by transabdominal ultrasound (aIMT) is an intermediate phenotype of cardiovascular risk. We aimed to (1) investigate the reproducibility of aIMT in a population-derived cohort of infants; (2) establish the distribution of aIMT in early infancy; (3) compare measurement by edge-detection software to that by manual sonographic calipers; and (4) assess the effect of individual and environmental variables on image quality. Methods Participants were term infants recruited to a population-derived birth cohort study. Transabdominal ultrasound was performed at six weeks of age by one of two trained operators. Thirty participants had ultrasounds performed by both operators on the same day. Data were collected on environmental (infant sleeping, presence of a sibling, use of sucrose, timing during study visit) and individual (post-conception age, weight, gender) variables. Two readers assessed image quality and measured aIMT by edge-detection software and a subset by manual sonographic calipers. Measurements were repeated by the same reader and between readers to obtain intra-observer and inter-observer reliability. Results Aortic IMT was measured successfully using edge-detection in 814 infants, and 290 of these infants also had aIMT measured using manual sonographic calipers. The intra-reader intraclass correlation (ICC) (n = 20) was 0.90 (95%CI 0.76, 0.96), mean difference 1.5 μm (95%LOA -39, 59). The between reader ICC using edge-detection (n = 20) was 0.92 (95%CI 0.82, 0.97) mean difference 2 μm (95%LOA -45.0, 49.0) and with manual caliper measurement (n = 290) the ICC was 0.84 (95%CI 0.80, 0.87) mean difference 5 μm (95%LOA -51.8, 61.8). Edge-detection measurements were greater than those from manual sonographic calipers (mean aIMT 618 μm (50) versus mean aIMT 563 μm (49) respectively; p < 0.001, mean difference 44 μm, 95%LOA -54, 142). With the exception of infant crying (p = 0.001), no associations were observed between individual and environmental variables and image quality. Conclusion In a population-derived cohort of term infants, aIMT measurement has a high level of intra and inter-reader reproducibility. Measurement of aIMT using edge-detection software gives higher inter-reader ICC than manual sonographic calipers. Image quality is not substantially affected by individual and environmental factors. [ABSTRACT FROM AUTHOR]