Intramyocellular lipids (IMCL) are depleted in response to an acute bout of exercise in lean endurance-trained individuals; however, it is unclear whether changes in IMCL content are also seen in response to acute and chronic exercise in obese individuals. We used magnetic resonance spectroscopy in 18 obese men and 5 normal-weight controls to assess IMCL content before and after an hour of cycling at the intensity corresponding with each participant’s maximal whole-body rate of fat oxidation (Fat_max). Fat_max was determined via indirect calorimetry during a graded exercise test on a cycle ergometer. The same outcome measures were reassessed in the obese group after a 16-week lifestyle intervention comprising dietary calorie restriction and exercise training. At baseline, IMCL content decreased in response to 1 h of cycling at Fat_max in controls (2.8 ± 0.4 to 2.0 ± 0.3 A.U., –39%, p = 0.02), but not in obese (5.4 ± 2.1 vs. 5.2 ± 2.2 A.U., p = 0.42). The lifestyle intervention lead to weight loss (–10.0 ± 5.4 kg, p < 0.001), improvements in maximal aerobic power (+5.2 ± 3.4 mL/(kg·min)), maximal fat oxidation rate (+0.19 ± 0.22 g/min), and a 29% decrease in homeostasis model assessment score (all p < 0.05). However, when the 1 h of cycling at Fat_max was repeated after the lifestyle intervention, there remained no observable change in IMCL (4.6 ± 1.8 vs. 4.6 ± 1.9 A.U., p = 0.92). In summary, there was no IMCL depletion in response to 1 h of cycling at moderate intensity either before or after the lifestyle intervention in obese men. An effective lifestyle intervention including moderate-intensity exercise training did not impact rate of utilisation of IMCL during acute exercise in obese men.