Abstract Background To test the effects of guided endurance training on work ability in middle-aged female hospital workers of various occupations. Methods We randomized 265 healthy, sedentary, middle-aged women (45–65 years) to an endurance training group (EG 210 min/week) or a wait-list control group (CG). At baseline and at 6-month follow-up, we assessed work ability (Work Ability Index [WAI]), physical activity (Freiburger activity questionnaire) and peak oxygen uptake (VO_2peak) by cardiopulmonary exercise testing. To examine the influence of baseline work ability, participants were divided into poor-moderate (WAI 1, 7–36 points, n = 83), good (WAI 2, 37–43 points, n = 136) and excellent (WAI 3, 44–49 points, n = 46) WAI subgroups. Results Cardiorespiratory fitness improved significantly after 6 months in the EG but not in the CG. The WAI total score increased significantly in the EG (38.3 ± 5.0 to 39.8 ± 4.9 points) but not in the CG (39.4 ± 4.7 to 39.3 ± 4.9 points), with a significant difference between groups (p < 0.01). In the EG, only the poor-moderate subgroup (WAI 1, 33.0 ± 2.9 to 36.6 ± 4.8 points, p < 0.05) increased the WAI total score, with this increase being significantly higher compared to the good (WAI 2, 40.2 ± 2.1 to, 40.4 ± 3.7 points) and excellent (WAI 3, 45.6 ± 1.5 to 45.7 ± 1.8 points) subgroup. Conclusions A 6-month guided exercise training intervention significantly increases cardiorespiratory fitness with concomitant improvements in work ability in middle-aged previously sedentary hospital employees. Women with low baseline work ability seem to particularly benefit from the intervention, which implies that similar interventions may be particularly beneficial for this group of individuals. Trial registration German Clinical Trails Register Identifier: DRKS00005159. Registered 25 September 2013.