Purpose: The purpose of this study was to investigate the face validity of the self-reported single item work ability with objectively measured heart rate reserve (%HRR) among blue-collar workers. Methods: We utilized data from 127 blue-collar workers (Female = 53; Male = 74) aged 18–65 years from the cross-sectional “New method for Objective Measurements of physical Activity in Daily living (NOMAD)” study. The workers reported their single item work ability and completed an aerobic capacity cycling test and objective measurements of heart rate reserve monitored with Actiheart for 3–4 days with a total of 5,810 h, including 2,640 working hours. Results: A significant moderate correlation between work ability and %HRR was observed among males (R = −0.33, P = 0.005), but not among females (R = 0.11, P = 0.431). In a gender-stratified multi-adjusted logistic regression analysis, males with high %HRR were more likely to report a reduced work ability compared to males with low %HRR [OR = 4.75, 95% confidence interval (95% CI) = 1.31 to 17.25]. However, this association was not found among females (OR = 0.26, 95% CI 0.03 to 2.16), and a significant interaction between work ability, %HRR and gender was observed (P = 0.03). Conclusions: The observed association between work ability and objectively measured %HRR over several days among male blue-collar workers supports the face validity of the single work ability item. It is a useful and valid measure of the relation between physical work demands and resources among male blue-collar workers. The contrasting association among females needs to be further investigated.