Background: Cardiorespiratory fitness is a strong predictor of all-cause mortality. Physical activity of at least moderate intensity can improve cardiorespiratory fitness. Workplaces may provide a relatively controlled setting in which to improve cardiorespiratory fitness through physical activity. Limited work has been conducted to quantify the impact of delivering physical activity in the workplace on cardiorespiratory fitness. Objective: The objective of this systematic review was to quantify the effects of workplace physical activity interventions on peak oxygen consumption (VO 2peak) and explore study and participant characteristics as putative moderators. Methods: Seven databases were searched up to September 2018. Search terms included “workplace”, “physical activity” and “intervention”. Inclusion criteria were controlled trials where physical activity of at least moderate intensity was delivered in the workplace and compared to controls or non-active comparators; and cardiorespiratory fitness measured by actual or predicted VO 2peak. Risk of bias was assessed using the PEDro scale. A random-effects meta-analysis was conducted with between-study variation quantified and then explored for putative predictors with a meta-regression. Pooled estimate uncertainty was expressed as 90% confidence intervals (CIs) and assessed against our threshold value for clinical relevance of 1 mL·kg −1·min −1. Results: The final dataset consisted of 25 estimates of VO 2peak from 12 trials. The pooled mean differences between intervention and control arms was a beneficial improvement of 2.7 mL·kg −1·min −1 (90% CI 1.6–3.8). The 95% prediction interval ranged from a reduction in VO 2peak of − 1.1 to an improvement of 6.5 mL·kg −1·min −1. Between-study heterogeneity (τau) was ± 1.6 mL·kg −1·min −1. The meta-regression showed longer interventions (3.2 mL·kg −1·min −1; 90% CI 1.6–3.8) to have an additive effect and studies with a low risk of bias (− 2.5 mL·kg −1·min −1; 90% CI − 4.0 to − 1.0), and participants of greater baseline VO 2peak (− 1.6 mL·kg −1·min −1; 90% CI − 3.6 to 0.4), and age (− 1.4 mL·kg −1·min −1; 90% CI − 3.2 to 0.3) having a lesser effect. Participant sex (percentage female) had an additive effect on VO 2peak (0.4 mL·kg −1·min −1; 90% CI − 1.6 to 2.4). Conclusions: Workplace-based physical activity interventions consisting of at least moderate-intensity activity improve cardiorespiratory fitness. At the present time, we surmise that no single group of employees (e.g. older employees or less fit individuals) can be definitively identified as standing to benefit more from workplace physical activity interventions than others. This demonstrates the potential utility of workplace physical activity interventions for improving cardiorespiratory fitness in a broad range of healthy employees. Protocol registration: PROSPERO (registration number: 42017057498).