The 6-foot-9 fifth-seeded Isner was beaten by fellow American Jesse Levine 6-3, 6-1.
Two other seeds advanced as No. 7 Canadian Frank Dancevic defeated American Taylor Dent 3-6, 7-5, 6-1, and No. 8 South African Kevin Anderson beat Luxembourg's Gilles Muller 6-7 (3-7), 6-3, 6-3. Dent, the 2002 Newport champion, was appearing in his first ATP-level match since February 2006.
The top seeds in Newport are American Mardy Fish and defending champion Fabrice Santoro, who defeated Nicolas Mahut in last year's all-French finale.
Notable deaths of 2014 [PHOTOS]