Background: Sedentary behavior (SB) and cardiorespiratory fitness (CRF) are important issues in occupational health. Developing a questionnaire to concurrently assess workers' SB and CRF could fundamentally improve epidemiological research. The Worker's Living Activity-time Questionnaire (WLAQ) was developed previously to assess workers' sitting time. WLAQ can be modified to evaluate workers' CRF if additional physical activity (PA) data such as PA frequency, duration, and intensity are collected. Methods: A total of 198 working adults (93 women and 105 men; age, 30-60 years) completed anthropometric measurements, a treadmill exercise test for measuring maximal oxygen consumption (VO 2max), and modified WLAQ (m-WLAQ, which included questions about PA data additional to the original questions). Multiple regression analyses were performed to develop prediction equations for VO 2max. The generated models were cross-validated using the predicted residual error sum of squares method. Among the participants, the data of 97 participants who completed m-WLAQ twice after a 1-week interval were used to calculate intraclass correlation coefficient (ICC) for the test-retest reliability analyses. Results: Age (r = − 0.29), sex (r = 0.48), body mass index (BMI, r = − 0.20), total sitting time (r = − 0.15), and PA score (total points for PA data, r = 0.47) were significantly correlated with VO 2max. The models that included age, sex, and BMI accounted for 43% of the variance in measured VO 2max [standard error of the estimate (SEE) = 5.04 ml•kg − 1 •min − 1 ]. These percentages increased to 59% when the PA score was included in the models (SEE = 4.29 ml•kg − 1 •min − 1). Cross-validation analyses demonstrated good stability of the VO 2max prediction models, while systematic underestimation and overestimation of VO 2max were observed in individuals with high and low fitness, respectively. The ICC of the PA score was 0.87 (0.82-0.91), indicating excellent reliability. Conclusions: The PA score obtained using m-WLAQ, rather than sitting time, correlated well with measured VO 2max. The equation model that included the PA score as well as age, sex, and BMI had a favorable validity for estimating VO 2max. Thus, m-WLAQ can be a useful questionnaire to concurrently assess workers' SB and CRF, which makes it a reasonable resource for future epidemiological surveys on occupational health.