Cygnus, loaded with two tons of trash, was released by the space station's robotic arm at 1330 GMT, NASA said.
Once the spacecraft is far enough from the station, some five hours after its release, NASA engineers will light a fire inside a special onboard module to study the flame's propagation and oxygen consumption in microgravity.
Experiments with fire have been rarely conducted on orbiting spacecraft because of the risk to crews on manned missions.
"NASA's objective is to reduce the risk of long-duration exploration missions, and a spacecraft fire is one of the biggest concerns for NASA and the international space exploration community," said Jason Crusan, director of NASA's advanced exploration systems director.
Cygnus also is to release five nano satellites known as "LEMUR CubeSats" that will form part of a constellation of satellites tracking maritime traffic and weather.
Cygnus will remain in orbit until Wednesday June 22, when its orbital engines will be activated in a series of maneuvers to cause it to plunge into the atmosphere and disintegrate over the Pacific.
The research in microgravity will be relayed back to Earth where the findings will help in preparing a manned mission to Mars.
Cygnus was launched March 22 aboard an Atlas V rocket from Cape Canaveral by the company United Launch Alliance.
It was Orbital ATK's fifth resupply mission to ISS for NASA.
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