Objectives This study aims to investigate across subgroups of healthcare workers (1) the changes in psychosocial working conditions and emotional exhaustion during the pandemic compared with the situation before, and (2) the impact of different stages of the COVID-19 pandemic in terms of hospital pressure on psychosocial working conditions and emotional exhaustion. Methods Five questionnaire measurements during 2 years from 1915 healthcare workers in the longitudinal study € the Netherlands Working Conditions Survey-COVID-19' were used. At each measurement, three subgroups were defined: working with patients with COVID-19, working with other patients and not working with patients. For each measurement, hospital pressure was determined by number of hospitalisations per day. Linear mixed models were fitted to analyse differences across subgroups of healthcare workers. Results During COVID-19, psychosocial working conditions deteriorated among healthcare workers working with patients, in particular with patients with COVID-19, compared with healthcare workers not working with patients after correcting for the situation before COVID-19. No changes were observed for emotional exhaustion in any of the subgroups. An increasing hospital pressure improved job autonomy and reduced emotional demands among healthcare workers in COVID-19 wards, but had no influence on other psychosocial working conditions and emotional exhaustion. Conclusion Psychosocial working conditions deteriorated for healthcare workers working with (COVID-19) patients during the pandemic, while emotional exhaustion did not change among all groups of healthcare workers.