Background: Faecal immunochemical test (FIT)-based colorectal cancer screening requires successive rounds for maximum preventive effect. Advanced neoplasia can bleed intermittently and thus might be missed by single faecal sampling. Few studies have been done on two sample FIT (2-FIT) screening over multiple rounds. Therefore, we compared multiple rounds of one sample FIT (1-FIT) with 2-FIT screening with respect to participation, positive predictive value (PPV), diagnostic yield, and interval colorectal cancer. Methods: In this population-based study, a random selection of asymptomatic individuals aged 50–74 years in the Rotterdam-Rijnmond region, Netherlands, were invited by post for four rounds (every 2 years) of 1-FIT or 2-FIT screening. Key exclusion criteria were a history or colorectal cancer or inflammatory bowel disease, colon imaging in the previous 2 years, and life expectancy of less than 5 years. Per round, invitees received one or two FITs to sample either one or two consecutive bowel movements. OC-Sensor Micro (Eiken Chemical Co., Ltd, Japan) FITs were used by all participants, except the fourth round of screening for the 1-FIT cohort, for which participants used either an OC-Sensor or a FOB-Gold (Sentinel Diagnostics, Milan, Italy). A faecal haemoglobin cutoff concentration of 10 μg/g of faeces in at least one test was used for referral for colonoscopy. Findings: Between 2006 and 2015, of 10 008 invited individuals for the 1-FIT cohort, 9787 were eligible for inclusion, of whom 7310 participated at least once in four successive rounds. Of 3197 invited individuals for the 2-FIT cohort, 3131 were eligible for inclusion, and 2269 participated at least once in four successive rounds. In the 1-FIT screening cohort, 74·7% (7310 of 9787) of invitees participated at least once versus 72·5% (2269 of 3131) of invitees in the 2-FIT cohort (p=0·013). Among participants who participated at least once, the cumulative positivity rate over four rounds was 19·2% (1407 of 7310) for the 1-FIT cohort versus 28·5% (647 of 2269) for the 2-FIT cohort (p<0·0001). The cumulative PPV for advanced neoplasia was 33·0% (432 of 1308 colonoscopies) for the 1-FIT cohort versus 24·2% (147 of 607 colonoscopies) for the 2-FIT cohort (p<0·0001). The cumulative diagnostic yield of advanced neoplasia among invited individuals was 4·4% (432 of 9787) for 1-FIT versus 4·7% (147 of 3131) for 2-FIT screening (p=0·46)). FIT interval colorectal cancers were detected in eight (0·1%) of 7310 participants in the 1-FIT cohort and two (0·1%) of 2269 with 2-FIT screening (p=1·00). Interpretation: Four rounds of 2-FIT screening with a low faecal haemoglobin cutoff level did not result in a significant increase in diagnostic yield or a decrease in interval colorectal cancers compared with 1-FIT, despite higher colonoscopy demand. Therefore, 1-FIT colorectal cancer screening programmes should be preferred. Funding: None.