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After Jonny Bairstow made 64 and Alex Hales 51, the tourists slipped from 116-1 to 211-9 – both Eoin Morgan and Jos Buttler were out first ball.
They needed last-wicket pair Adil Rashid and David Willey to reach 282.
India A coasted in the chase, Ajinkya Rahane’s 91 getting them home with more than 10 overs to spare.
This setback comes after England beat a different India A line-up by three wickets on Tuesday.
It also throws up further questions over the top-order batting.
The regular top four would comprise Hales, Jason Roy – who was unlucky to be bowled when a part of his helmet fell on to the bails – Joe Root and Morgan.
Root has not played in the warm-ups after arriving late because of the birth of his son, while captain Morgan, returning to the side after missing the tour of Bangladesh over security fears, has made only three runs in two innings.
Meanwhile, Sam Billings made 93 in the first match and Bairstow pressed his claim here.
Morgan is not the only man short of form. Moeen Ali has made just one run in his two innings and was the third of three wickets to fall in the space of eight balls.
Rashid, who shared 71 for the 10th wicket with Willey, served up a succession of short balls in seven overs of leg spin that went for 51 – and not one of England’s bowlers managed an economy rate of under six.