Cyclic stretch of differentiated myotubes mimics the loading pattern of mature skeletal muscle. We tested a cell culture model of disuse atrophy by the cessation of repetitive bouts of cyclic stretch in differentiated C2C12 myotubes. Myotubes were subjected to cyclic strain (12%, 0.7 Hz, 1 h/d) on collagen-I-coated Bioflex plates using a computer-controlled vacuum stretch apparatus (Flexcell Int.) for 2 (2dSTR) or 5 (5dSTR) consecutive days. Control cultures were maintained in the Bioflex plates without cyclic stretch for 2 d or 5 d. Additionally, some cultures were stretched for 2 d followed by cessation of stretch for 3 d (2dSTR3dCES). Cyclic stretching (5dSTR) increased myotube diameter and overall myotube area by similar to 2-fold (P < 0.05) compared to non-stretched controls, while cessation of stretch (2dSTR3dCES) resulted in similar to 80% smaller myotubes than 5dSTR cells, and 40-50% smaller than non-stretched controls (P < 0.05). Further, the calpain-dependent cleavage products of alpha II-spectrin (150 kDa) and talin increased (3.5-fold and 2.2-fold, respectively; P < 0.05) in 2dSTR3dCES myotubes, compared to non-stretched controls. The I h cyclic stretching protocol acutely increased the phosphorylation of Ala (+4.5-fold; P < 0.05) and its downstream targets, FOXO3a (+4.2-fold; P < 0.05) and GSM-3 beta (+1.8-fold; Pc 0.05), which returned to baseline by 48 h after cessation of stretch. Additionally, nitric oxide production increased during stretch and co-treatment with the NOS inhibitor, L-NAME, inhibited the effects of stretch and cessation of stretch. We conclude that cessation of cyclic stretching causes myotube atrophy by activating calpains and decreasing activation of Akt. Stretch-induced myotube growth, as well as activation of atrophy signaling with cessation of stretch, are dependent on NOS activity. (C) 2013 Elsevier Inc. All rights reserved.