Germany's Kaymer, who tied the course record with a sizzling nine-under-par 63 on Thursday, followed up with a second-round 69 to post a 12-under total of 132 in the PGA Tour's flagship event widely regarded as the unofficial fifth major.
Young gun Spieth, at 20 one of the most exciting prospects in the game, fired a superb bogey-free 66 to occupy second place with compatriot Russell Henley (71) a further three strokes back at eight under.
Breezy weather on another hot day at the TPC Sawgrass made the par-72 Stadium Course a more difficult prospect for the players, and Kaymer was delighted to remain at the top after mixing five birdies with two bogeys.
"I didn't hit as many fairways as yesterday but I managed my way quite nicely around the golf course," the 29-year-old from Dusseldorf told reporters after covering the back nine in two-under 34.
"I had a couple of nice up-and-downs and two longer putts went in, so I kept it really well together. Overall, 12 under par. I'm very pleased.
"It was a little difficult today. I thought it was already gusty in the morning, and the pin positions were a little bit tougher than yesterday."
Kaymer has not won a tournament since the Nedbank Golf Challenge in South Africa at the end of 2012, but has gained confidence from his improving form over the past month.
"I have had a lot of good finishes," said the former world number one. "The way I have played golf, I am very happy over the last four or five weeks especially. The next step is just putting yourself in contention, and hopefully win.”
LEAD TRIMMED
Kaymer, whose only PGA Tour victory came at the highest level with his playoff win at the 2010 PGA Championship, birdied two of the last four holes to seize an early five-stroke lead before that was trimmed by Spieth in the afternoon.
"Solid round from tee to green," said Spieth, the 2013 PGA Tour rookie of the year who tied for second at last month's Masters where he was bidding to eclipse Tiger Woods by becoming the event's youngest champion.
"I felt like today was one of my best ball-striking days I've had in a long time. Very stress-free, and I'd like to have a round like that the next two days."
Tour veteran Jim Furyk was at six under after carding a 68, level with fellow American Gary Woodland, U.S. Open champion Justin Rose, former world number one Lee Westwood and Spaniard Sergio Garcia who all posted 71s.
Australian world number two Adam Scott, one of four players competing at the Players Championship who could dislodge the absent Woods from the top spot after Sunday's final round, made the cut right on the number after carding a 67.
Woods, who won this event last year, is continuing his recovery from back surgery.
Scott, who needs to finish no worse than joint 16th this week to become number one for the first time, rebounded from a dismal opening 77 to finish at even-par 144, level with Northern Ireland's Rory McIlroy (74).
McIlroy appeared likely to miss the cut after staggering to the turn in a woeful six-over 42 but he birdied four of the last seven holes, sinking a 12-footer on the 18th green, to qualify for the third round.
"It's funny, I feel very comfortable on the back nine here," McIlroy said after coming home in four-under 32. "If I could only just figure out the front nine, I would be okay."
British Open champion Phil Mickelson, a five-times major winner, missed the cut for a second consecutive year.
(Reporting by Mark Lamport-Stokes; Editing by Gene Cherry)
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