Once in court she told the judge she would "rather starve to death than to change my opinions in this regard", the Washington Post reported.
Manning, who was born Bradley Manning, stole and leaked a massive cache of military reports and State Department intelligence to WikiLeaks, which has been identified by USA intelligence agencies as a front for Russian government operations.
Manning has said she objects to grand juries in principle.
Manning spent 62 days in the Alexandria City Jail before being released last week after the term of that grand jury expired. He also imposed fines that will kick in at $500 a day after 30 days and $1,000 a day after 60 days.
The heavy-handed punishment betrays the growing frustration of federal prosecutors, for who this is a second attempt at making Manning testify. The subpoena "represents an improper and impermissible use of the grand jury process", the spokesperson said.
The US Justice Department has asked Britain to extradite Assange to stand trial in the United States for "conspiracy" for advising Manning on breaking into a restricted US government computer.
"There's nothing new", Manning said Thursday during a press conference before her court hearing in Alexandria.
Manning was released because the previous subpoena expired.
Manning's attorney, Moira Meltzer-Cohen, addressed the media after Manning was returned to jail, expressing her disappointment and reiterating that her client would not betray her convictions by testifying. "It is telling that the United States has always been more concerned with the disclosure of those [leaked] documents than with their damning substance", she told reporters.
"There's nothing dishonourable in discharging your responsibility as a U.S. citizen", Judge Trenga told her. She said the isolation caused her "extraordinary pain" and that she was sometimes in a "dissociative stupor". Obama commuted Manning's sentence just days before leaving office.
Prosecutors are seeking Manning's testimony for its case against WikiLeaks founder Julian Assange. She was charged with leaking only portions of a couple of hundred documents and was sentenced by a military judge to 35 years in prison in 2013.
"The questions are the same questions I was asked before the court-martial", Manning said.
We "never once made this about anything other than obtaining immunized testimony in furtherance of an investigation", he said, adding that "all we want is her to truthfully answer questions as our constitutional republic requires of our citizens".