
The 6ft 10in (2.08m) Isner was beaten by 6ft 11in (2.11m) fellow American Reilly Opelka 7-6 (7/4), 7-6 (8/6), 6-7 (4/7), 7-6 (7/5) in a battle of the giants.
No fewer than 87 aces were powered down between the big-serving pair with just one break apiece being registered in the fourth set of a match that lasted 2hr 58min.
Isner, 33, had not lost a Grand Slam match to a player ranked as low as number 97 Opelka, who is 12 years his junior, since retiring due to a foot injury while trailing 106th-ranked Martin Klizan in the first round at Melbourne in 2014.
Opelka has never before reached the second round of a Slam and will next face Italy’s world number 102 Thomas Fabbiano, who beat Australian wildcard Jason Kubler 6-4, 7-6 (7/1), 2-6, 6-3.