Background: The decline in postabsorptive and postprandial muscle protein fractional synthesis rates (FSR) does not quantitatively account for muscle atrophy during uncomplicated, short-term disuse, when atrophy rates are the highest. We sought to determine whether 2 days of unilateral knee immobilization affects mixed muscle protein fractional breakdown rates (FBR) during postabsorptive and simulated postprandial conditions. Methods: Twenty-three healthy, male participants (age: 22 ± 1 year; height: 179 ± 1 cm; body mass: 73.4 ± 1.5 kg; body mass index 22.8 ± 0.5 kg·m−2) took part in this randomized, controlled study. After 48 h of unilateral knee immobilization, primed continuous intravenous l-[15N]-phenylalanine and l-[ring-2H5]-phenylalanine infusions were used for parallel determinations of FBR and FSR, respectively, in a postabsorptive (saline infusion; FAST) or simulated postprandial state (67.5 mg·kg body mass−1·h−1 amino acid infusion; FED). Bilateral m. vastus lateralis biopsies from the control (CON) and immobilized (IMM) legs, and arterialized-venous blood samples, were collected throughout. Results: Amino acid infusion rapidly increased plasma phenylalanine (59 ± 9%), leucine (76 ± 5%), isoleucine (109 ± 7%) and valine (42 ± 4%) concentrations in FED only (all P < 0.001), which was sustained for the remainder of infusion. Serum insulin concentrations peaked at 21.8 ± 2.2 mU·L−1 at 15 min in FED only (P < 0.001) and were 60% greater in FED than FAST (P < 0.01). Immobilization did not influence FBR in either FAST (CON: 0.150 ± 0.018; IMM: 0.143 ± 0.017%·h−1) or FED (CON: 0.134 ± 0.012; IMM: 0.160 ± 0.018%·h−1; all effects P > 0.05). However, immobilization decreased FSR (P < 0.05) in both FAST (0.071 ± 0.004 vs. 0.086 ± 0.007%·h−1; IMM vs CON, respectively) and FED (0.066 ± 0.016 vs. 0.119 ± 0.016%·h−1; IMM vs CON, respectively). Consequently, immobilization decreased net muscle protein balance (P < 0.05) and to a greater extent in FED (CON: −0.012 ± 0.025; IMM: −0.095 ± 0.023%·h−1; P < 0.05) than FAST (CON: −0.064 ± 0.020; IMM: −0.072 ± 0.017%·h−1). Conclusions: We conclude that merely 2 days of leg immobilization does not modulate postabsorptive and simulated postprandial muscle protein breakdown rates. Instead, under these conditions the muscle negative muscle protein balance associated with brief periods of experimental disuse is driven near exclusively by reduced basal muscle protein synthesis rates and anabolic resistance to amino acid administration.