Abstract Objective Demographic changes encompass societies to maintain the work ability (WA) of aging workforces. The present study explored the relationship between modifiable lifestyle factors, cognitive functions, and their influence on WA, using a multi-group structural equation approach. Method Cross-sectional data from 247 middle-aged and 236 older employees from the Dortmund Vital Study were included in this analysis. We proposed a model with three exogenous variables (Physical Fitness, Cognitive Functions, and Social Life), and with WA as the endogenous variable. WA was measured with the Work Ability Index (WAI), which considers job demands and individual physical and mental resources. Multi-group analyses were based on the principles of invariance testing and conducted using robust estimation methods. Results Results revealed that Social Life outside work had significant positive effects on WA in both, middle-aged and older adults. Physical Fitness had a significant effect on WA only in middle-aged adult, and Cognitive Functions had no significant influence on WA in either group. In older adults, Physical Fitness correlated with Cognitive Functions, whereas in middle-aged adults, Cognitive Functions marginally correlated with Social Life. Conclusions Our results underline the importance of an active social life outside the workplace for WA, regardless of the employees’ age. The influence of Physical Fitness on WA changes with increasing age, indicating the necessity to have a differentiated view of age effects and interacting influencing factors. Our research contributes to the knowledge of how WA could be most effectively promoted in different age groups. Clinicaltrials.gov NCT05155397; https://clinicaltrials.gov/ct2/show/NCT05155397.