Purpose: Peak oxygen uptake (VO2peak) is a strong predictor of cardiovascular and all-cause mortality risk. Physical activity interventions such as walking are commonly used to improve cardiometabolic health in sedentary populations. However, walking may not provide a sufficient stimulus to improve VO2peak in those with a higher baseline fitness. Thus, the purpose of this study was to examine changes in VO2peak in insufficiently active adults that were classified based on baseline cardiorespiratory fitness (< 8 METS or ≥ 8 METS) following a six-month walking intervention. We hypothesized that those in the <8 MET group would see a greater improvement in VO2peak when compared to those in the ≥8 MET group. Methods: Cross-sectional, secondary analyses were carried out on data from a clinical trial that included insufficiently active, mostly overweight and obese adults (n= 520). Subjects underwent treadmill-based graded exercise testing (modified Balke protocol) with ventilatory gas exchange analyses to determine VO2peak. Valid VO2peak tests were defined as subjects achieving both >90% of their age predicted max heart rate and an RER > 1.0. Two hundred and one subjects met our prespecified criteria (Males/Females: 69/132, Age: 45.4 ± 9.3 years, BMI: 32.2 ± 6.2 kg/m2, VO2peak: 25.1 ± 5.0 mL/kg/min). Subjects were then placed into 2 fitness categories based on baseline VO2peak: < 8 METS (<28 ml/kg/min) and ≥8 METS (≥28 ml/kg/min). Linear mixed models were used to assess fixed effects on VO2peak (time and baseline fitness category) as well as the interaction of time ✕ fitness category. Data are presented as mean ± SD and α was set at 0.05. Results: One hundred and forty five subjects were in the <8 MET group (M/F: 33/112, Age: 46.6 ± 6.3, BMI: 33.3 ± 6.3) and fifty six in the ≥8 METs group (M/F: 36/30, Age: 42.2 ± 9.6, BMI: 28.9 ± 5.0). No significant improvements were seen in VO2peak in response to the intervention (pre: 25.1 ± 4.8; post: 25.2 ± 5.1, p = 0.36). When the cohort was divided into the above specified fitness groups, a significant group by time interaction was found between the < 8 MET group (pre: 22.8 ± 3.0, post: 23.1 ± 3.6 ml/kg/min) and ≥8 MET group (pre: 31.1 ± 3.1, post: 30.5 ± 4.5 ml/kg/min) ( p = 0.038). While no changes were seen in VO2peak in response to the intervention, a significant correlation was observed between change in VO2peak and change in moderate to vigorous physical activity (mins/days) (ρ = 0.254, p < 0.001). Conclusions: Our secondary data analyses showed that there was no difference in VO2peak following the walking intervention, however there was a significant group by time interaction found between fitness groups. While statistically significant, the changes are likely not clinically relevant. These data suggest that increasing physical activity alone, with no intensity guidance, may be insufficient to increase cardiorespiratory fitness in those with already healthy fitness l Supported by NIH R01CA198915 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.