[2] The test involves placing a fecal sample on guaiac paper (containing a phenolic compound, alpha-guaiaconic acid, extracted from the wood resin of Guaiacum trees) and applying hydrogen peroxide which, in the presence of blood, yields a blue reaction product within seconds.

[citation needed] After applying the feces, one or two drops of hydrogen peroxide are then dripped on to the other side of the film, and it is observed for a rapid blue color change.

[citation needed] When the hydrogen peroxide is dripped on to the guaiac paper, it oxidizes the alpha-guaiaconic acid to a blue colored quinone.

This is particularly common if the recommended dietary preparation is not followed, as the heme in red meat or the peroxidase or catalase activity in vegetables, especially if uncooked, can cause analytical false positives.

[19] The package insert guidelines from the manufacturers, for example Hemoccult SENSA,[20] recommend that nonsteroidal anti-inflammatory drugs (NSAID), such as ibuprofen and aspirin, and iron supplements be discontinued for at least several days before the tests.

There is a concern that these agents may irritate the body and cause biologically positive tests even in the absence of a more substantial illness,[21][22] but there is some doubt about how frequently this occurs with NSAID medication.

The US Multisociety Task Force (MSTF) looked at 6 studies that compared high sensitivity gFOBT (Hemoccult SENSA) to FIT, and concluded that there were no clear difference in overall performance between these methods,[31] and a similar recommendation was made by the National Guideline Clearinghouse (NGC).

[34] The Centers for Disease Control and Prevention (CDC) in a 2006–2007 survey found extensive inappropriate use of low sensitivity gFOBT and of single specimens; it is unclear if these widespread suboptimal approaches have since declined.

[37] However, although heme breakdown is less than globin during intestinal transit, false negative results can be seen with the stool guaiac tests due to degradation of the peroxidase-activity.