Spanish national of Palestinian origin Saif Abu Keshek and Brazilian Thiago Avila were among dozens of activists aboard a flotilla intercepted in international waters off the coast of Greece on Thursday.
The two were seized by Israeli forces and brought to Israel for questioning, while the others were taken to the Greek island of Crete and released.
On Tuesday, an Israeli court extended the detention of Abu Keshek and Avila until Sunday to allow police more time to interrogate them, according to their lawyers.
The pair's legal team then filed an appeal at the Beersheva district court against the detention.
But their appeal was rejected.
"Today, the district court of Beersheva denied our appeal and basically accepted all of the arguments that the state or the police have represented before the court and kept the previous decision," Hadeel Abu Salih said, referring to the earlier court's ruling.
The two activists had appeared in the district court, their feet shackled, an AFP journalist reported.
Abu Keshek looked exhausted and sat with his hands clasped in his lap, while Avila seemed calm, said the journalist.
Israeli rights group Adalah, which is representing the pair, has called their detention illegal.
"As we said from the first day, we're talking about an illegal arrest that took place in international waters where the activists were kidnapped by the Israeli navy without any authority," Abu Salih said after the hearing on Wednesday.
"It's so concerning that also the legal system is giving a free hand for the Israeli forces to continue with this illegal arrest in a way that would give it also legitimacy to do it again and again and kidnap international nationals."
'Stopped drinking'
Adalah has also accused the authorities of subjecting the men to continuous abuse in detention.
"Saif basically told us that he stopped drinking water in addition to the hunger strike he started on Thursday morning," Abu Salih said.
"They are also telling us that they keep interrogating them for most of the time, most of the day, and the questions keep going around the same context, which is the humanitarian mission context."
Adalah had earlier said that the two men were being held in "total isolation, subjected to 24/7 high-intensity lighting in their cells and kept blindfolded whenever they were moved, including during medical examinations".
Israeli authorities have rejected the allegations but have filed no charges against the men.
Adalah said authorities have accused the pair of several offences, including "assisting the enemy during wartime" and "membership in and providing services to a terrorist organisation".
Israel's foreign ministry says both men were affiliated with the Popular Conference for Palestinians Abroad (PCPA), a group accused by Washington of "clandestinely acting on behalf of" Palestinian militant group Hamas.
Spain, Brazil and the United Nations have called for their swift release.
"It is not a crime to show solidarity and attempt to bring humanitarian aid to the Palestinian population in Gaza, who are in dire need of it," UN rights office spokesman Thameen Al-Kheetan said in a statement.
The flotilla had set sail from France, Spain and Italy with the aim of breaking Israel's blockade of Gaza and delivering humanitarian aid to the war-ravaged Palestinian territory.
Israel controls all entry points into Gaza, which has been under an Israeli blockade since 2007.
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