Astronauts Piers Sellers and Mike Fossum floated into Discovery's payload bay to paste a sealant on pre-damaged heat shield samples to test the material's performance in zero gravity.
During the seven hour spacewalk, the astronauts used a caulk gun and spatula to cover the samples with the material, whose texture they compared to peanut butter, soft putty and mud.
In the spacewalk's first task, Sellers stood at the tip of the International Space Station's robotic arm to record images of Discovery's leading wing edges with an infrared camera.
NASA extended the mission by a day, to 13 days, in order to squeeze in the third spacewalk.
In February 2003, the space shuttle Columbia's thermal protection was cracked by a piece of foam insulation that peeled off its external fuel tank during liftoff, causing the shuttle to break into a ball of fire as it returned to Earth.
Since then, NASA has made modifications to the massive orange fuel tank to reduce the amount of debris it loses during launch, and has tested in-orbit repair techniques in the two shuttle missions since the Columbia accident.
After examining hundreds of images of Discovery's heat shield, the US space agency declared the shuttle free of damage that could endanger its return to Earth on Monday.