An imbalance between physical work demands and physical capacity of the worker may be a risk factor for poor health. Perceived physical exertion provides information about the individual perception of the work demands relative to the capacity to perform the work. This study estimates the risk for long-term sickness absence (LTSA) from perceived physical exertion among healthcare workers. This prospective cohort study comprises 8592 Danish healthcare workers who responded to a baseline questionnaire in 2004-2005 and subsequently were followed for one year in the Danish Register for Evaluation of Marginalization (DREAM), a national register of social transfer payments. Using Cox regression hazard ratio (HR) analysis, controlled for age, gender, body mass index (BMI), smoking, tenure, leisure-time physical activity, psychosocial working conditions, and LTSA during one year prior to baseline, we modeled risk estimates of moderate and strenuous (reference: light) perceived physical exertion during healthcare work for onset of LTSA (receiving sickness absence compensation for ≥8 consecutive weeks) during 1-year follow-up. At baseline, 35.1%, 39.4%, and 25.5% of the healthcare workers experienced, respectively, light, moderate, and strenuous physical exertion during healthcare work. During follow-up, the 12-month prevalence of LTSA was 4.6%, 6.4%, and 8.9%, respectively, in these three exertion groups. A dose-response pattern between physical exertion and the risk for LTSA was found (trend test P<0.0001). In the multi-adjusted model, the risk for LTSA was 1.31 [95% confidence interval (95% CI) 1.04-1.64] for healthcare workers reporting moderate physical exertion and 1.57 (95% CI 1.23-2.01) for those reporting strenuous physical exertion, referencing those reporting light physical exertion during healthcare work. Moderate and strenuous perceived physical exertion during healthcare work increases - in a dose-response manner - the risk for LTSA. The possible preventive effect of balancing work demands with the capacity of the worker, to thereby avoid excessive physical exertion, should be tested in randomized controlled trials.