This study assessed the associations of (1) within-individual improvements and (2) within-individual deteriorations in working conditions, health behaviour and body mass index (BMI) with changes in work ability and self-rated health among workers. Prospective cohort study. The Netherlands. Persons in paid employment, aged 45-64 years, who participated in the Dutch Study on Transitions in Employment, Ability and Motivation (STREAM) between 2010 and 2017, and improved or deteriorated at least once with respect to working conditions (psychological and emotional job demands, autonomy, social support, physical workload), health behaviour (moderate and vigorous physical activity, smoking status), or BMI between any of two consecutive measurements during the 7-year follow-up. Changes in self-reported work ability on a scale from 0 to 10 (1st item of the work ability index) and self-rated health on a scale from 1 to 5 (SF-12). Of the 21 856 STREAM participants, ultimately 14 159 workers were included in the fixed effects analyses on improvements (N=14 045) and deteriorations (N=14 066). Workers with deteriorated working conditions decreased in work ability (β's: -0.21 (95% CI: -0.25 to -0.18) to -0.28 (95% CI: -0.33 to -0.24)) and health (β's: -0.07 (95% CI: -0.09 to -0.06) to -0.10 (95% CI: -0.12 to -0.08)), whereas improvements were to a lesser extent associated with increased work ability (β's: 0.06 (95% CI: 0.02 to 0.09) to 0.11 (95% CI: 0.06 to 0.16)) and health (β's: 0.02 (95% CI: 0.00 to 0.03) to 0.04 (95% CI: 0.02 to 0.06)). Workers with increased BMI or decreased physical activity reduced in work ability and health. Likewise, decreased BMI or increased vigorous physical activity was associated with improved health. An increase in moderate or vigorous physical activity was modestly associated with a reduced work ability. Quitting smoking was associated with reduced work ability and health. Compared with improvements, preventing deteriorations in working conditions, health behaviour and BMI, might be more beneficial for work ability and workers' health. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.