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Introduction The impact of cycling at different cadences on cardiopulmonary exercise test (CPET) measurements is poorly understood, especially when looking at parameters that are relevant in the clinical setting. We aimed to investigate whether higher cadences of pedalling led to meaningful changes in physiological endpoints meaning cycling protocols could be more flexible to patients.Methods Study participants were recruited from healthy staff members working within three NHS trusts across England. At baseline, all participants completed a CPET at 60 rpm, then subsequently completed CPETs at cadences of 70, 80, and 90 rpm, allocated in a random order. To evaluate the mean differences in CPET measurements across the cadences, we used a one-way repeated measures ANOVA. We then performed post-hoc pairwise comparisons with Tukey correction to account for multiple testing.Results Data collection took place between the 19th of September 2023 and 9th of April 2024. 25 participants had complete data at each cadence. 48% (12 of 25) were female, with a median (IQR) age of 30 years (27–41). There was no significant difference in peak &Vdot;O2 across the cadences. Maximum achieved work rate was significantly different across the cadences (p=0.0001). The highest wattage was achieved at 60 rpm (221.2 watts ±71.4) and lowest at 90 rpm (210.4 watts, ±77.2). End-exercise ventilation increased with increasing cadence (p=0.0129), with a mean of 97.6 L/min (±28.3) at 60 prm and 107.0 L/min (±33.9) at 90 prm. There was a significant difference in the cardiovascular slope (p=0.011), with mean cardiovascular slope 48.5 (±13.1) at 60 rpm and 52.4 (±16.8) at 90 rpm. Finally, there was a small but significant difference in test duration across the four cadences (P=<0.001), with the shortest duration (9.4 minutes) seen at 90 rpm and the longest (10.1 minutes) at 60 rpm. There were minimal differences in other CPET parameters.Conclusion In a healthy population, cycling cadence has little impact on estimates of overall cardiorespiratory fitness. However, higher cycling cadences appear to increase ventilatory demand and heartrate response to exercise and reduce maximum achieved work rate. This could have implications for CPETs in the clinical setting, where physiological responses to higher cadences may be more exaggerated.