Protein oxidation may play a role in the balance between anabolism and catabolism. We assessed the effect of a protein restricted diet on protein oxidation as a possible reflection of whole body protein metabolism. Sixteen healthy males (23 ± 3 years) were instructed to use a 4-day isocaloric protein restricted diet (0.25 g protein/kg body weight/day). Their habitual dietary intake was assessed by a 4-day food diary. After an overnight fast, a 30 g 13C-milk protein test drink was administered, followed by 330 min breath sample collection. Protein oxidation was measured by Isotope Ratio Mass Spectrometry. To assess actual change in protein intake from 24-h urea excretion, 24-h urine was collected. During the 4-day protein restricted diet, the urinary urea:creatinine ratio decreased by 56 ± 9%, which is comparable to a protein intake of ~0.65 g protein/kg body weight/day. After the protein restricted diet, 30.5 ± 7.3% of the 30 g 13C-milk protein was oxidized over 330 min, compared to 31.5 ± 6.4% (NS) after the subject's habitual diet (1.3 ± 0.3 g protein/kg body weight/day). A large range in the effect of the diet on protein oxidation (-43.2% vs. +44.0%) was observed. The residual standard deviation of the measurements was very small (0.601 ± 0.167). This suggests that in healthy males, protein oxidation is unaffected after a protein restricted diet. It is uncertain how important the role of fluctuations in short-term protein oxidation is within whole body protein metabolism.