Changes in skeletal muscle mass are controlled by mechanisms that dictate protein synthesis or degradation. The current human study explored whether changes in activation of the phosphoinositide 3-kinase (PI3K)-Akt1, p38, myostatin, and mRNA expression of markers of protein degradation and synthesis occur soon after withdrawal of weight bearing. Biopsies of the vastus lateralis muscle (VL) and soleus muscle (Sol) were obtained from eight healthy men before and following 3 days of unilateral lower limb suspension (ULLS). Akt1, Forkhead box class O (FOXO)-1A, FOXO-3A, p38, and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) phosphorylation and protein levels and myostatin protein level were analyzed by Western blot. Levels of mRNA of IGF1, FOXO-1A, FOXO-3A, atrogin-1, MuRF-1, caspase-3, calpain-2, calpain-3, 4E-BP1, and myostatin were measured using real-time PCR. The amounts of phosphorylated Akt1, FOXO-1A, FOXO-3A, and p38 were unaltered (P>0.05) after ULLS. Similarly, mRNA levels of IGF1, FOXO-1A, FOXO-3A, caspase-3, calpain-2, and calpain-3 showed no changes (P>0.05). The mRNA levels of atrogin-1 and MuRF-1, as well as the mRNA and protein phosphorylation of 4E-BP1, increased (P<0.05) in VL but not in Sol. Both muscles showed increased (P<0.05) myostatin mRNA and protein following ULLS. These results suggest that pathways other than PI3K-Akt stimulate atrogin-1 and MuRF-1 expression within 3 days of ULLS. Alternatively, transient changes in these pathways occurred in the early phase of ULLS. The increased myostatin mRNA and protein expression also indicate that multiple processes are involved in the early phase of muscle wasting. Further, the reported difference in gene expression pattern across muscles suggests that mechanisms regulating protein content in human skeletal muscle are influenced by phenotype and/or function.