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Objectives: This study aimed to estimate and compare the prevalence of the virus-specific antibodies against the SARS-CoV-2 nucleoprotein antigen (anti-SARS-CoV-2 N) in healthcare workers and an all-comer paediatric and adult patient population., Design, Setting and Participants: A longitudinal study enrolling healthcare professionals and concurrent serial cross-sectional studies of unselected all-comer patients were conducted at an Austrian academic medical centre. Healthcare workers were tested at enrolment and after 1, 2, 3, 6 and 12 months. The cross-sectional studies in patients were conducted at three time periods, which roughly coincided with the times after the first, second and third wave of SARS-CoV-2 in Austria (ie, 24 August-7 September 2020; 8-22 February 2021 and 9-23 November 2021). Anti-SARS-CoV-2 N antibodies were measured using a sandwich electrochemiluminescence assay (Roche)., Results: In total, 2735 and 9275 samples were measured in 812 healthcare workers (median age: 40 years, 78% female) and 8451 patients (median age: 55 years, 52% female), respectively. Over the entire study period, anti-SARS-CoV-2 N antibodies were detected in 98 of 812 healthcare workers, resulting in a seroprevalence of 12.1% (95% CI 10.0% to 14.5%), which did not differ significantly (p=0.63) from that of the all-comer patient population at the end of the study period (407/3184; 12.8%, 95% CI 11.7% to 14.0%). The seroprevalence between healthcare workers and patients did not differ significantly at any time and was 1.5-fold to 2-fold higher than the number of confirmed cases in Austria throughout the pandemic. In particular, there was no significant difference in the seroprevalence between paediatric and adult patients at any of the tested time periods., Conclusion: Throughout the pandemic, healthcare staff and an adult and paediatric all-comer patient population had similar exposure to SARS-CoV-2., Trial Registration Number: ClinicalTrials.gov Identifier: NCT04407429., Competing Interests: Competing interests: MR, CN, CJB, ESS, TS, FJ, EA, FH, HH, TP and CH report no conflicts of interest for this work. Outside of the submitted work, CJB reports honoraria for serving on advisory boards and speaker honoraria from Amgen, Daiichi-Sankyo, Novartis, SOBI; board member of Technoclone. ESS reports research grants from the US National Health Institutes (NIH), the European Commission’s Program Horizon 2020, the Austrian Science Funds (FWF) and the Austrian Research Promotion Agency (FFG). HH reports grants from Glock Health Science and Research, BlueSky Immunotherapies and the Austrian Ministry of Education Science and Research. TAZ reports research grants from the Austrian Science Funds and the German Research Foundation, honoraria for serving on advisory boards from Boehringer Ingelheim, personal fees from Alkem Metabolics, AstraZeneca, Boehringer Ingelheim and Sun Pharmaceutical Industries and educational grants from Eli Lilly and Company., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)