The duo hit a combined 86 aces in the delayed third-round tussle, meaning rallies and break points were at a premium until Lopez made the decisive break in the 11th game of the fourth set before securing a 6-7(8) 7-6(6) 7-6(3) 7-5 victory in two hours 51 minutes.
"No, it's not fun," said Lopez when asked if he enjoyed the match. "I mean it's fun because I won it, but it was a difficult match to play.
"When you beat players like John and go through a difficult match like this, you have to be proud of yourself. There is no rhythm; a lot of aces, a lot of points where you don't play. You have to always be aware that a chance is coming."
Isner, whose exit left the United States without a representative in the last 16 of the men's or women's singles for the first time since 1911, blasted 52 aces and snatched the first-set tiebreak before the Spaniard levelled with some crunching forehands in the second.
Play was suspended briefly when rain fell on Court Three and when the pair returned it was Lopez who claimed the third tiebreak.
Then, just as the crowd were expecting to see another set go all the way, 19th seed Lopez found a way past Isner's defences, punching the air in celebration, before holding serve and sealing victory with yet another ace.
"Even though you know where he (Isner) is going to serve, sometimes it's really difficult,” said Lopez, who retained his Aegon International grasscourt title at Eastbourne this month.
"I was guessing. Sometimes I make it; sometimes I don't."
The Spaniard next meets Australian Open champion Stanislas Wawrinka in the fourth round and could face a busy few days after another rain-affected day in southwest London.
"I know it's going to be a busy week if I keep winning," he said, adding that he will have to play very well against a "confident" Wawrinka.
(Editing by Tony Jimenez and David Goodman)
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