BACKGROUND: There is evidence that physical exercise training (PET) conducted at the workplace is effective in improving physical fitness and thus health. However, there is no current systematic review available that provides high-level evidence regarding the effects of PET on physical fitness in the workforce. OBJECTIVES: To quantify sex-, age-, and occupation type-specific effects of PET on physical fitness and to characterize dose-response relationships of PET modalities that could maximize gains in physical fitness in the working population. DATA SOURCES: A computerized systematic literature search was conducted in the databases PubMed and Cochrane Library (2000-2019) to identify articles related to PET in workers. STUDY ELIGIBILITY CRITERIA: Only randomized controlled trials with a passive control group were included if they investigated the effects of PET programs in workers and tested at least one fitness measure. STUDY APPRAISAL AND SYNTHESIS METHODS: Weighted mean standardised mean differences (SMD(wm)) were calculated using random effects models. A multivariate random effects meta-regression was computed to explain the influence of key training modalities (e.g., training frequency, session duration, intensity) on the effectiveness of PET on measures of physical fitness. Further, subgroup univariate analyses were computed for each training modality. Additionally, methodological quality of the included studies was rated with the help of the Physiotherapy Evidence Database (PEDro) Scale. RESULTS: Overall, 3423 workers aged 30-56 years participated in 17 studies (19 articles) that were eligible for inclusion. Methodological quality of the included studies was moderate with a median PEDro score of 6. Our analyses revealed significant, small-sized effects of PET on cardiorespiratory fitness (CRF), muscular endurance, and muscle power (0.29 ≤ SMD(wm) ≤ 0.48). Medium effects were found for CRF and muscular endurance in younger workers (≤ 45 years) (SMD(wm) = 0.71) and white-collar workers (SMD(wm) = 0.60), respectively. Multivariate random effects meta-regression for CRF revealed that none of the examined training modalities predicted the effects of PET on CRF (R(2) = 0). Independently computed subgroup analyses showed significant PET effects on CRF when conducted for 9-12 weeks (SMD(wm) = 0.31) and for 17-20 weeks (SMD(wm) = 0.74). CONCLUSIONS: PET effects on physical fitness in healthy workers are moderated by age (CRF) and occupation type (muscular endurance). Further, independently computed subgroup analyses indicated that the training period of the PET programs may play an important role in improving CRF in workers.