ImportanceDisentangling the effects of different SARS-CoV-2 variants and of vaccination on the occurrence of post-acute sequelae of SARS-CoV-2 (PASC) is crucial to estimate and potentially reduce the future burden of PASC. ObjectiveTo determine the association of primary SARS-CoV-2 infection on the frequency of PASC symptoms by viral variant and vaccination status. DesignCross-sectional questionnaire and SARS-CoV-2 serology (May/June 2022) performed within a prospective healthcare worker cohort (SURPRISE study). SettingMulticenter study in nine healthcare networks from North-Eastern Switzerland. ParticipantsVolunteer sample of healthcare workers (HCW) from participating institutions. Of approximately 20000 eligible participants, 3870 registered for the cohort and 2912 were included in this analysis. ExposuresSARS-CoV-2 infection documented by positive nasopharyngeal swab (>4 weeks ago), stratified by viral variant and vaccination status at time of infection, compared to absence of documented infection (no positive swab, negative serology). Main OutcomeSum score of eighteen self-reported PASC symptoms. ResultsAmong 2912 participants (median age 44 years, 81.3% female), SARS-CoV-2 infection was reported by 1685 (55.9%) participants, thereof 315 (18.7%) during Wild-type, 288 (17.1%) during Alpha/Delta, and 1082 (64.2%) during Omicron circulation. Mean symptom number in previously infected participants significantly exceeded that of uninfected controls (0.39), but decreased with recency of the viral variant 1.12 (p<0.001) for Wild-type (median time since infection 18.5 months), 0.67 (p<0.001) for Alpha/Delta (6.6 months), and 0.52 (p=0.005) for Omicron BA.1 (3.1 months) infected participants. After Omicron BA.1 infection, the mean symptom score was 0.49 (p=0.30) for those with [≥]3 prior vaccinations and 0.71 (p=0.028) with 1-2 previous vaccinations compared to 0.36 for unvaccinated individuals. Adjusting for confounders, Wild-type (adjusted risk ratio [aRR] 2.81, 95% confidence interval [CI] 2.08-3.83) and Alpha/Delta infection (aRR 1.93, 95% CI 1.10-3.46) showed significant associations with the outcome, whereas Omicron BA.1 infection (aRR 1.29, 95% CI 0.69-2.43) and vaccination before infection (aRR 1.27, 95% CI 0.82-1.94) did not. Conclusions and RelevancePrevious infection with pre-Omicron variants was the strongest risk factor for reporting PASC symptoms in this HCW cohort. A definite influence of prior vaccination on the prevention of PASC after Omicron BA.1 infection was not measurable.