On a day where many riders impressed, Marquez made his much-anticipated Ducati debut and gelled straight away with the GP23.
The eight-time world champion made a strong start as he jumped up to third after his opening stint.
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But it was after lunch that Marquez really put on a show as he went fastest with a low 1m 29s lap.
Marquez then improved further before Vinales, who was consistently fast, went back atop the leaderboard.
Also enjoying a strong showing with his new team was MotoGP rookie Pedro Acosta, as the GASGAS Tech 3 KTM rider was just 1.2 seconds down on the top time before a late fall at turn two.
Two-time MotoGP champion Francesco Bagnaia had a solid outing as tested the new GP24 bike, as did Jorge Martin although the Pramac rider suffered two falls. The first of those came very soon after jumping onto the new Ducati bike.
Bringing a new RC213V to the fore, Honda had a strong day as Luca Marini found good pace as the test went on.
Joan Mir was a constant presence around the top ten as well, while Johann Zarco enjoyed a decent showing aside from a slip off in the morning.
Providing a late charge was Brad Binder who moved up to second, but after setting another red sector to begin his next lap, the KTM rider then crashed at turn five after entering the corner slightly off-line.
Marco Bezzecchi had a strong test and finished as the top Ducati ahead of Marc Marquez.
The same was also true for Raul Fernandez, who managed to rival Vinales for pace at different stages of the one-day test.