An attenuated ability to appropriately oxidize fat (metabolic inflexibility) has been associated with the development of obesity and type 2 diabetes. Previous studies have found that regular exercise training increases the body’s ability to oxidize fat during exercise, but also shown that fat oxidation at the same relative and absolute exercise intensity is lower in old compared with young adults. Based on these studies we investigated the effect of training status on the whole-body peak fat oxidation rate (PFO) during exercise in young and middle-aged trained and untrained men. We hypothesized that aging was associated with decreased PFO, but regular exercise training could counteract this decline. 36 healthy non-overweight young and middle-aged men were recruited into a four groups: young (27 [24–30] yrs, (Mean [95% CI])) untrained (⩒O2peak: 47 [44–49] ml/min/kg), young (28 [26–30] yrs) trained (⩒O2peak: 64 [62–67] ml/min/kg), middle-aged (55 [53–57] yrs) untrained (⩒O2peak: 37 [32–42] ml/min/kg) and middle-aged (54 [51–57] yrs) trained (⩒O2peak: 55 [51–58] ml/min/kg). PFO was measured by indirect calorimetry while subjects performed a validated incremental exercise protocol on a cycle ergometer. Whole-body peak fat oxidation rate was higher in the young trained compared to young untrained subjects (0.70 [0.65–0.75] vs.0.45 [0.36–0.54] g/min, post-hoc: p < 0.001); however, this training effect was attenuated in middle-aged trained and untrained subjects (0.44 [0.38–0.50] vs. 0.41 [0.35–0.47] g/min, post-hoc: p = 0.83, respectively). In summary, these findings suggest that the training induced effects on whole-body fat oxidation found in young men may be attenuated in middle-aged men.