Little basic science data exists regarding the thickness of transected stomach as the limits of smaller gastric sleeves are created closer to the lesser curvature in laparoscopic sleeve gastrectomy (SG). We sought to determine the tissue thickness trends along the staple line and examine what factors might predispose to thicker tissue. This was a single-center, single-surgeon nonrandomized prospective study of patients undergoing SG. Excised SG specimens, with patient consent, underwent tissue measurement at multiple predetermined locations and at the midpoint of each fired staple cartridge. After 9 months, ending in July 2012, we met our goal enrollment of 50 gastric sleeve specimens. Most of the patients were female (80 %) and white (92.5 %). Average age was 42 years (range, 19-60 years), and average body mass index (BMI) was 49 kg/m(2) (range, 34-82 kg/m(2)). Tissue thickness was significantly different (p < 0.01) at each location, with the antrum being the thickest at 2.70 mm, followed by the midbody at 2.33 mm, and the fundus at 1.97 mm. Both male gender (+0.32 mm, p = 0.04) and BMI over 50 kg/m(2) (+0.33 mm, p < 0.01) were associated with thicker tissue, but only in the antrum. The most significant transitions in thickness occurred on the fourth and fifth staple fires, dropping 0.24 mm (p = 0.02) and 0.35 mm (p < 0.01), respectively. Tissue thickness of excised SG specimens varies on the basis of location with the antrum being the thickest. Both BMI (>50 kg/m(2)) and gender (male) are associated with increased tissue thickness, but only in the antrum. Surgeons should consider using a thicker staple load, such as black, when these factors are present. Also, significant changes in tissue thickness at the fourth and fifth staple fires suggest stepwise alteration in staple cartridge color selection.