The aim of the present study was to investigate the influence of fixation delay and the perioperative ischemia on hypoxia inducible factor (HIF)-1α gene expression, HIF-1α protein expression, and immunohistochemical (IHC) expression of HIF-1α, GLUT-1, Bcl-2, and Ki-67 in colorectal cancer. The study included 25 surgically removed colorectal tumors. Three sets of samples were collected readily after removal and exposed to 0, 30, and 60 minutes of delay of fixation or freezing. The perioperative ischemia time was registered. In each set of the samples, HIF-1α gene expression was analyzed by quantitative real time polymerase chain reaction, protein concentration of HIF-1α was assessed by enzyme-linked immunosorbent assay, and IHC staining of HIF-1α, GLUT-1, Bcl-2, and Ki-67 was performed. Preoperative formalin-fixed paraffin-embedded biopsies and whole sections of the entire tumor were analyzed by IHC. We found that the HIF-1α gene expression, HIF-1α protein concentration, and IHC expression of HIF-1α, GLUT-1, Ki-67, and Bcl-2 were not systematically affected by either the fixation or freezing delay of the tissue, the perioperative ischemia time, or the total ischemia time (perioperative ischemia+delay of fixation or freezing) in colorectal tumors. However, the intraindividual variation was quite large, which may question the use of individually, non-standardized-handled single biopsies or small tissue samples for analysis of often rather heterogenously expressed biomarkers.