Maverick Vinales led Andrea Iannnone by 0.121 seconds in MotoGP FP2 at Assen after a late surge on his Yamaha, while FP1 pacesetter Marquez was eighth.
A rise in temperatures for the 45 minutes of afternoon running saw most switching their attention to race preparation, though Marquez was able to set the best lap of the day so far early on with a 1:34.041s.
The order behind his reshuffled itself over the next 15 minutes, before Ducati's Andrea Dovizioso edged 0.002s ahead of the Honda rider with a 1:34.039s, with Suzuki's Alex Rins a further 0.006s adrift in third.
Much of the 24-strong field opted for a fresh slicks towards the end of the session for a qualifying simulation, though Marquez remained on his hard/hard combination through to the chequered flag.
Vinales was the first big mover in the order at the end, taking over top spot with a 1:33.547s on his opening gambit, and improved to a 1:33.378s on his penultimate tour which stood as the best of the session.
Suzuki's Iannone used the updated engine his GSX-RR is running to good effect to end the day second, 0.121s adrift of Vinales, while Pramac's Danilo Petrucci completed the top three as top Ducati man.
Valentino Rossi was a few thousandths shy of his compatriot in fourth on the sister factory YZR-M1, while Cal Crutchlow took top Honda honours on his LCR machine with a late 1:33.812s to go fifth.
Dovizioso found himself shuffled back to sixth as the dust settled, with Ducati team-mate Jorge Lorenzo, who spent much of the afternoon evaluating the soft/soft combination he used to dominate in Barcelona last time out, just behind in seventh.
Marquez's desire to complete his race run on the harder tyres means he was eighth at the chequered flag, with Suzuki's Alex Rins leaping up from 15th to ninth in FP2.
Johann Zarco crashed at Turn 9 in the final quarter of the session, but was able to get back out on his second Tech3 M1 at the end to secure the final provisional Q2 spot in 10th, denying the second factory Honda of Dani Pedrosa 0.034s.
KTM's Pol Espargaro also took a visit to the Assen gravel trap 15 minutes in, the Spaniard coming off at Turn 7. He ended the session down in 19th, while teammate Bradley Smith was 18th after encountering early technical troubles on his RC16.
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