Security officials quickly encircled Novak Djokovic and Andy Murray during a changeover in the second set with the Serbian world number one leading 7-6 (7/5), 4-3.
A white banner unfurled over advertising hoardings on the side of the court on Rod Laver Arena read "Australian Open for Refugees #shutdownmanus."
A woman was carried from the court by security officials with at least four others, wearing Australian Open Refugee t-shirts, led from the grandstands before play resumed after five minutes.
Several dignitaries were in the crowd, including tennis legends Margaret Court and Rod Laver and Australian Hollywood stars Toni Collette and Eric Bana.
Australia has adopted a hardline policy against asylum-seekers arriving by boat, which has frequently sparked criticism.
Since July 2013 the government has sent boatpeople to camps on Papua New Guinea's Manus Island and Nauru in the Pacific.
They are denied resettlement in Australia even if they are found to be genuine refugees.
The policy is designed to stop the flow of boatpeople heading to Australia, who had been arriving almost daily in often unsafe wooden fishing vessels, with hundreds drowning en route.
Refugee advocates claimed several of them swallowed razor blades and washing powder before the protest was broken after several days.
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