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There is few research on the causal pathways in the link between obesity and work ability. Furthermore, there are indications that the effects of obesity on work ability differ for workers with different levels of physical workload. This study aimed to investigate the moderating effect of physical workload in the relation between obesity and work ability.A longitudinal study was conducted among 36,435 Dutch construction workers who participated in at least two periodic medical examinations during the years 2008–2015. Logistic regression analyses were used to investigate the effect of manual material handling and strenuous work postures on the association between obesity and work ability. Confounding effects were tested for age, educational level, smoking, vigorous physical activity, psychosocial work demands, and working hours. Additive interaction between obesity and physical workload on work ability was tested using the relative excess risk due to interaction (RERI).Construction workers with overweight (OR=1.09; 95% CI: 1.02 to 1.16) or obesity (OR=1.27; 95% CI: 1.17 to 1.38) had an increased risk of poor/moderate work ability. Exposure to manual material handling (OR=1.58; 95% CI: 1.49 to 1.68) or strenuous work postures (OR=1.80; 95% CI: 1.70 to 1.90) also increased the risk of poor/moderate work ability. The effect of the combination of obesity with high physical workload was greater than the sum of the individual effects (strenuous work postures: RERI=0.39; 95% CI: 0.10 to 0.67; manual material handling: RERI=0.26; 95% CI: 0.02 to 0.51).Obesity and high physical workload were associated with poor work ability and had a synergistic, negative effect on work ability. To prevent poor work ability, there is a need for interventions that promote a healthy lifestyle and increase physical capacity, as well as for interventions that tailor physical workload to the individual physical capacity.