Endurance athletes frequently employ nutritional strategies to enhance performance. While professional organizations recommend high carbohydrate (HC) diets to maximize performance, many athletes, and researchers have recently shown renewed interest in the ketogenic diet (KD) in hopes to promote “fat adaptation”, which would allow athletes to make use of the essentially unlimited energy resources from stored body fat. This would circumvent one fatigue mechanism, the depletion of muscle glycogen stores, that has been considered central to performance outcomes in endurance events. The present study investigated the effects of participants’ habitual diet (HD), HC, and KD on endurance performance in a 30-km simulated cycling time trial (TT), physiological responses during the TT, and muscle session fuel percentile (SFP) before and after the TT using ultrasonic imaging. Due to the COVID-19 pandemic, data collection ceased after only six recreational cyclists and triathletes (f = 4, m = 6; age: 37.2 ± 12.2; VO2max: 46.8 ± 6.8 ml/kg/min; weekly cycling distance: 225.3 ± 64.2 km). Due to the small sample size, we do not report inferential statistics for our primary outcome measure, cycling performance. Participants completed the KD at the lowest power output. Oxygen consumption (V̇O2), heart rate (HR), and perceived exertion (RPE) during the TT were similar in all conditions. FATox rates were highest in the KD condition and lowest in the HC condition. SFP was lower during KD compared with HD and lower following the TT compared with fasted resting values across all conditions. We discuss methodological considerations into the use of exercise equipment, nutritional interventions, and statistical analysis strategies for study designs like the present. Further research is needed to assess the impact of HC and KD on TT performance in this population. ClinicalTrials.gov Identifier: NCT04097171; OSF preregistration: https://osf.io/ujx6e/