Background: The aim of this study was to analyze the expression of hypoxia-inducible factor (HIF)-1α during ischemia and after reperfusion in muscle tissue in the context of microsurgical free muscle tissue transfer. Methods: Ten patients with soft-tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischemia in normoxia, another one was taken after 72 ± 11 minutes of ischemia, and the last one was taken 77 ± 22 minutes after reperfusion. The samples were analyzed using DNA microarray, real-time polymerase chain reaction, and immunohistochemistry. Results: DNA microarray, real-time polymerase chain reaction, and immunohistochemistry did not provide evidence of differential expression of HIF-1α comparing ischemia and reperfusion to normoxia. However, DNA microarray showed an up-regulation of activating transcription factor-3 during ischemia and spermine N1-acetyltransferase-1 during ischemia and reperfusion. Conclusions: This study shows that ischemia and reperfusion induce alterations on the gene expression level in human muscle free flaps. Data from this study indicate that the expression of HIF-1α might not be affected but that other putative pathways of ischemic regulation might be of great interest. Finally, these findings correspond with the surgeon's clinical experience that the accepted times of ischemia, generally up to 90 minutes, are not sufficient to induce pathophysiologic processes, which can ultimately lead to flap loss.