The Spain international started alongside Tiago and Gabi as Diego Simeone's men ruthlessly took apart the Liga pacesetters, but he only lasted 10 minutes before being replaced by goalscorer Saul.
Koke was out for a week in January after hurting his hamstring, though he could now be set for a longer spell out after picking up what looks to be a similar problem.
The 23-year-old is reportedly facing up to a month on the sidelines.
The injury served as a disappointing blow at the end of a marvellous day for Atleti, although Saul could now be handed a chance to build on his fine performance in the derby should Koke's absence be confirmed.
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