Having bowled out England for 283 earlier in the morning, India looked in charge at 148-2 at tea before Adil Rashid’s double strike jolted the hosts who lost three wickets in 19 balls to slump to 156-5.
Ashwin enhanced his new-found batting reputation with an unbeaten 57 while all-rounder Ravindra Jadeja was batting on 31 at stumps with India trailing by 12 runs.
Paceman Mohammed Shami claimed the last two English wickets earlier in the morning as the tourists added 15 runs to their overnight score before being dismissed for 283.
Murali Vijay walked out with Parthiv Patel, his third opening partner of the series, to lead India’s reply and it turned out to be an eventful yet brief stay for the right-hander.
Vijay survived an obstructing-the-field appeal, was dropped by Jos Buttler and the batsman eventually walked off, nicking a harmless Ben Stokes delivery even though umpire Chris Gaffaney did not spot the nick.
Patel, by contrast, looked determined to make the most of his chance return to the Indian side after eight years in the wilderness.
The diminutive 31-year-old, replacing injured regular wicketkeeper Wriddhiman Saha, smacked six boundaries in his 42 before Adil Rashid dismissed him leg-before.
Pujara and Kohli then joined forces to frustrate the tourists with the steady accumulation of 75 runs for the third wicket.
Dropped on 35 by Jonny Bairstow, Pujara went on to make 51 before falling to a Rashid long-hop and his dismissal sparked off a middle order collapse.
Rashid, in his next over, trapped the scoreless Ajinkya Rahane leg-before with a googly and Karun Nair’s Test debut was soured after his almighty mix-up with Kohli.
Buttler was outstanding at backward point, diving to stop the ball and then throwing down the stumps to send back Nair for four.
Kohli showed tremendous restraint for major part of his knock, especially as England attacked him with a heavily-packed off-side, but he eventually fell to a soft dismissal, edging Stokes behind the wicket. Kohli’s 62 included nine boundaries.
Ashwin hit eight boundaries in his unbeaten knock, having raised 67 runs with Jadeja for the seventh wicket.