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Athletic Bilbao captain Iker Muniain fired his side into the lead in the opening two minutes at the San Mames in the round-of-16 clash.
Ferran Torres, who arrived in January from Manchester City in a £46.3m deal, pulled Barcelona back into the lead with a superb strike 18 minutes later.
Inigo Martinez struck late for Bilbao in the second half before Pedri pulled back an injury-time equaliser to send the Copa del Rey tie to extra-time.
Fati came off the bench in the second half to replace Ferran Jutgla, but the Barcelona wonderkid left the pitch early in the first half of extra-time.
The 19-year-old forward recently returned for Barcelona from a hamstring injury after missing several matches in late 2021.
The exact moment where Ansu Fati was injured again. 💔😔— BARÇA WORLD (@BarcaWorld99) January 20, 2022
Ansu Fati heads down the tunnel with an injury. pic.twitter.com/yhJ4yokXT2— ESPN FC (@ESPNFC) January 20, 2022
Fati was in tears after picking up his thigh injury against Bilbao and immediately walked down the tunnel after his exit from the pitch.
Muniain ensured Barcelona crashed out of the Copa del Rey after sending Marc-Andre ter Stegen the wrong way from the spot-kick in the first half of extra-time.
Speaking after the 3-2 defeat, Barcelona manager Xavi revealed that Fati had been affected by an injury and the club would wait to learn more details.
“Tomorrow we will see [about the extent of the injury],” he told reporters.
“He has been affected, but tomorrow we will see the diagnosis.”
Xavi also confirmed that Barcelona trio Jordi Alba, Gerard Pique and Pedri had picked up individual knocks.
Barcelona on Thursday confirmed that Fati has suffered a left biceps femoris injury and the La Liga club are waiting to hear more details after tests.
“[Ansu Fati] has a left biceps femoris injury,” Barcelona tweeted. “Further tests pending to find out the exact extent of the injury.”