Experts have warned against the pandemic burden on healthcare workers early on, however little is known about the evolution of this burden with time, in addition to the long-term effects of post-COVID symptoms in healthcare workers. Staff at the Geneva University Hospitals in Switzerland had an online follow-up in July and December 2021, on their physical and mental health, quality of life and functional capacity using validated scales. Descriptive analyses compared the prevalence of symptoms, functional impairment and quality of life in SARS-CoV-2 positive and negative individuals at baseline and at follow-up. Out of the initial n = 3,083 participants that answered at baseline in July 2021, n = 900 (mean age of 46.4 years, 70.1% women) completed the follow-up in December 2021. With time, more individuals reported fatigue (+ 9.4%), headache (+ 9.0%), insomnia (+ 2.3%), cognitive impairment (+ 1.4%), stress/burnout (+ 8.8%), pain (+ 8.3%), digestive symptoms (+ 3.6%), dyspnea (+ 1.0%), and cough (+ 7.7%) compared to baseline, with a differentially larger increase in symptoms in the SARS-CoV-2 negative group. Individuals had more functional impairment (12.7% at baseline and 23.9% at follow-up), with more absenteeism and worsening quality of life. Healthcare workers are potentially suffering from long term consequences of the pandemic burden, calling for urgent action and solutions.