The aim of this study was to assess the response of key mTORC1 substrates to a bout of contractile stimuli under different times of functional unloading. Functional unloading of hind-limb muscles was carried out by the method of antiorthostatic suspension. Twenty-eight Wistar rats were divided into four groups: control, and hindlimb suspension for 1, 3, and 7 days. After hindlimb suspension, isolated soleus muscles of rats were subjected to a bout of ex vivo eccentric contractions. The contents of phosphorylated forms of p70s6k and 4E-BP1 were then determined using western blotting. It was found that an eccentric load resulted in a significant increase in p70s6k phosphorylation and reduced 4E-BP1 phosphorylation both in control and suspended rats, but in the case of suspension the response was dramatically reduced. Thus, it can be concluded that a bout of eccentric contractions of isolated rat soleus muscle during functional unloading causes a weaker activation of the Akt-mTORC1-p70s6k signaling pathway compared with the control. This may indicate that it is important to maintain muscle tone for a more efficient muscle perception of an external mechanical signal and subsequent activation of anabolic signaling pathways.