Captain Michael Vaughan and batsman Kevin Pietersen have both suffered injuries with six days to go before England's first Test in India.
Vaughan was ruled out of the final warm-up game, which began on Thursday, with a recurrence of knee trouble.
And Pietersen retired hurt in the middle of the day with back trouble.
Vaughan had surgery in December to clear the knee problem, which forced an early return from Pakistan, but felt soreness after last week's game.
Marcus Trescothick, who did the job in Vaughan's absence late last year, took over as captain.
"We remain hopeful he will be fit for next week's Test," said a spokesman.
"Michael has experienced soreness in his right knee since the first practice match. This has gradually got worse.
"He tested the knee at the nets this morning and it was decided as a precautionary measure to rule him out of the three-day game."
Pietersen hit an unbeaten 47 before being helped from the field.
He showed signs of discomfort after lunch but continued at the crease after physio Kirk Russell applied a back support and strapping to the problem area.
A pulled six caused him to grimace in pain, however, and he retreated to the dressing room five minutes later.
With Paul Collingwood ruled out of the match with back spasms, the tourists' batting resources are now under strain.
That is not the only concern for England as a stomach bug, which has swept through the tour party, kept Simon Jones and Shaun Udal out of the game against an Indian Board President's XI.
Vaughan, 31, was ruled out of the first Test in Pakistan and missed the one-day series there because of the same injury, which has hit continually over the last few years.
But he was passed fit to travel to India after his operation went well and only 11 days ago insisted he felt "very positive" about the state of his knee. But it has been heavily strapped during practice sessions and coach Duncan Fletcher revealed his concern when he said: "I think we can look to it maybe being a recurring injury for the rest of his career."