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Fecal immunochemical test (FIT) screening for colorectal cancer (CRC) requires timely successive rounds for an optimal preventive effect. However, data on attendance and trend in yield over multiple rounds of FIT screening are limited. We therefore conducted a consecutive third round of FIT screening in a population-based CRC screening trial.Average-risk subjects aged 50–74 years were approached for three rounds of 1-sample FIT (OC-sensor) screening. Subjects with a hemoglobin level ≥50 ng/ml (≥10 μg Hb/g) feces were referred for colonoscopy. Subjects with a positive FIT in previous rounds were not re-invited for FIT screening.In the first round, 7,501 subjects were invited. The participation rate was 62.6% in the first round, 63.2% in the second round, and 68.3% in the third round (P<0.001). In total, 73% (5,241/7,229) of all eligible subjects participated in at least one of three rounds. The positivity rate was significantly higher in the first (8.4%) round compared with the second (6.0%) and third (5.7%) screening rounds (P<0.001). The detection rate of advanced neoplasia (AN) declined from the first round to subsequent rounds (round 1: 3.3%; round 2: 1.9%; and round 3: 1.3%;P<0.001). The positive predictive value for AN was 40.7% in the first screening round, 33.2% in the second screening round, and 24.0% in the third screening round (P<0.001).Repeated biennial FIT screening is acceptable with increased participation in successive screening rounds, and >70% of all eligible subjects participating at least once over three rounds. The decline in screen-detected AN over three screening rounds is compatible with a decreased prevalence of AN as a result of repeated FIT screening. These findings provide strong evidence for the effectiveness of FIT screening and stress the importance of ongoing research over multiple screening rounds.