Recently, there has been interest in whether shift work may enhance infection susceptibility. Our aim was to determine whether shift workers in healthcare have a higher incidence, duration, and severity of influenza-like illness (ILI) and acute respiratory illness (ARI) than non-shift workers. From September 2016-June 2017, 501 rotating and/or night shift workers and 88 non-shift workers from the Klokwerk+ study (2016-2017) daily registered occurrence of ILI/ARI symptoms using a smartphone application. Incidence rate of ILI/ARI episodes (defined as ≥2 symptoms on the same day/≥1 symptom during two consecutive days), mean duration per episode, and incidence rate of severe episodes were compared between shift and non-shift workers using negative binomial regression and linear mixed model analysis. In total, participants completed 110,347 diaries. Shift workers' incidence rate of ILI/ARI episodes was 1.20 (95%-CI:1.01, 1.43) times higher, and for severe ILI/ARI episodes 1.22 (95%-CI:1.01, 1.49) times higher, compared to non-shift workers. Mean duration per ILI/ARI episode did not differ (ratio between means=1.02 (95%-CI:0.87, 1.19)). In conclusion, shift workers in healthcare had more and more severe ILI/ARI episodes than non-shift workers, but with a similar duration. Insight into underlying mechanisms connecting shift work and infection susceptibility will contribute to the design of preventive initiatives.