Messi had been feeling pain in his hamstring and despite coach Jorge Sampaoli insisting on Monday the striker was fit, he did not even make the bench at the Wanda Metropolitano in Madrid.
After staying an unused substitute in a 2-0 win over Italy on Friday, Messi’s absence will do little to ease concerns about the 30-year-old’s condition.
He has played 47 matches already in all competitions this season.
Barcelona travel to Sevilla on Saturday in La Liga before hosting Roma four days later in the first leg of the Champions League quarter-finals.
Gonzalo Higuain was picked up front instead for Argentina as Sampaoli’s side looked to deliver a morale-boosting performance against Spain ahead of this summer’s World Cup in Russia.
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