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Riding a prototype of Honda's 2018 bike, Marquez, three days after securing a fourth world title, posted a time of 1min 30.033sec on the Ricardo-Tormo circuit near Valencia.
His Spanish compatriot, Maverick Vinales posted the second fastest time overall at 0.156sec having topped the charts on Tuesday.
His Yamaha team-mate Valentino Rossi, the nine-time world champion, was third overall with Marquez's Honda team-mate Dani Pedrosa fourth, having managed the second fastest time on Wednesday.