Three former athletes at the University of Pennsylvania women’s swimming program sued the school, the Ivy League Council of Presidents and athletics organizations Tuesday, claiming they violated federal law to allow transgender athlete Lia Thomas to compete against them.

The lawsuit comes amid a flurry of efforts attempting to keep transgender women from competing in women’s sports, including legal moves in all three branches of the federal government such as President Donald Trump signing an executive order to that effect.

The complaint filed by Grace Estabrook, Ellen Holmquist and Margot Kaczorowski focuses on the 2022 Ivy League women’s championships, where Thomas won four first-place medals and set multiple records, demanding that her records be vacated.

“The Ivy League engaged in a season-long pressure campaign to keep Thomas eligible to compete and prevent women from speaking up for their equal rights,” the lawsuit alleges.

In addition to UPenn and the Ivy League, the lawsuit names the NCAA and Harvard University, which hosted the 2022 league championship event, accusing them of violating Title IX, which prohibits sex discrimination in education programs and activities that receive federal funding. Thomas is not a defendant in the suit.

The plaintiffs are represented by attorney Bill Bock, a former NCAA Infractions Committee member who also filed suit last year on behalf of other collegiate swimmers in connection with Thomas’ participation in the 2022 NCAA championships.

“This lawsuit exposes the behind the scenes scheming that led to the attempt by Harvard University, UPenn, the Ivy League and the NCAA, to impose radical gender ideology on the American college sports landscape,” Bock said in a statement to CNN.

“College sports are the premier stage for women’s sports in America, and while the NCAA does not comment on pending litigation, the Association and its members will continue to promote Title IX, make unprecedented investments in women’s sports and ensure fair competition in all NCAA championships,” the NCAA said in a statement Thursday.

The NCAA also announced Thursday an overhaul of its transgender athlete policy to limit participation in women’s sports, which the organization said it was in response to Trump’s executive order.

“A student-athlete assigned male at birth may not compete on a women’s team,” the new policy states. It also prohibits transgender male athletes who receive testosterone treatment from competing on a women’s team.

“This national standard brings much needed clarity as we modernize college sports for today’s student-athletes,” NCAA President Charlie Baker said in a statement.

A spokesperson for Harvard said, “We don’t comment on active litigation.” CNN did not immediately receive a response Thursday to requests for comment from the other defendants. No hearings have yet been scheduled in the case.

League accused of favoritism toward Thomas

The plaintiffs say they were discouraged from expressing disagreement with the university’s stand to accommodate Thomas, who had previously competed on the UPenn men’s swimming team, including Thomas’ presence in women’s locker rooms.

A swim coach allegedly had told Kaczorowski that Thomas would not be using the same locker room when Thomas joined the women’s team, but that changed in the fall of 2021. That coach told Kaczorowski “there was nothing he could do” about it, according to the lawsuit.

“Harvard did not provide a unisex bathroom or separate bathroom for Thomas to use or for any other women to use who did not want to use the Women’s Locker room while Thomas was using it” during the Ivy League Championships, the complaint states.

The lawsuit also accuses Ivy League Executive Director Robin Harris of fighting against tighter NCAA rules on transgender participation in swimming, which the NCAA was considering and would have made Thomas ineligible to compete in 2022. Harris did not respond to a CNN request for comment Thursday.

Under NCAA guidelines adopted in 2010, athletes assigned male at birth could participate in women’s sports as long as they had been using testosterone suppression medication for at least a year, a standard that has been heavily scrutinized as to whether it potentially gives recently transitioned athletes an unfair physical advantage.

In 2022, the NCAA adopted new sport-specific standards requiring testing for most transgender athletes to assure their testosterone levels are below a defined level. The rules for swimming, announced less than a month before the 2022 women’s championships, would have made it impossible for Thomas to participate because it required three years of documented hormone levels, dating back to before her transition.

The association ultimately delayed implementation of the new standard until after the 2022 championship events, saying the short window for compliance “could have unfair and potentially detrimental impacts on schools and student-athletes.”

“The Ivy League had won when the NCAA backed down and changed its nationwide rules to bring Thomas back into compliance and renew Thomas’ eligibility,” the lawsuit states.

Plaintiff Ellen Holmquist did not qualify for the Ivy League Championships that year. Holmquist claims she was told by a coach that she had missed the cutoff by one spot, arguing that Thomas’ participation effectively denied her the opportunity to compete.

Transgender athletes facing backlash

Republicans now in control of the White House and both houses of Congress have quickly implemented their promised rollback of transgender participation in sports. Trump signed an executive order Wednesday cutting federal funding to educational programs in institutions that allow transgender athletes in women’s events.

“With this executive order, the war on women’s sports is over,” Trump said.

The House passed a similar bill last month. It has not yet been considered by the Senate.

The order was criticized by Athlete Ally, which advocates for transgender athletes, saying, “Our hearts break for the trans youth who will no longer be able to know the joy of playing sports as their full and authentic selves.”

Last year, the National Association of Intercollegiate Athletics, which organizes sports at 250 smaller colleges and universities, announced it was effectively banning athletic participation by transgender women in most of its women’s sports programs.

“Only NAIA student-athletes whose biological sex is female may participate in NAIA-sponsored female sports,” the association said, providing exceptions only for practices, team activities and exhibitions.

The latest legal moves are causing whiplash within collegiate compliance departments after the Biden administration previously introduced regulations stating Title IX protections apply to transgender people. But a proposal to make that guidance apply explicitly to women’s athletics was shelved, and the broader rules were overturned last month by a federal judge.

Lia Thomas has not commented publicly on the latest lawsuit. Despite her expressed intention to keep swimming competitively after college, Thomas has been barred from international events by the rules of World Aquatics, which only qualify transgender athletes who have not experienced biological puberty.

The Court of Arbitration for Sport denied Thomas’ challenge to the rule, making her ineligible for most elite competitions, including the 2024 Olympics.

CNN’s Jessie Yeung and Samantha Waldenberg contributed to this report.