Background: Health-related lifestyle behaviors are linked to many noncommunicable/chronic diseases. The 9-item Lifestyle Questionnaire is based on an adaptation of the Lifestyle Risk Scale and measures seven health-related behaviors: diet, alcohol intake, sleep deficit, physical activity, obesity, smoking, and exposure to secondhand smoke. This study aimed to characterize the test-retest reliability of the Lifestyle Questionnaire. Methods: The Lifestyle Questionnaire was administered at screening and baseline visits of a multicenter (42 sites), multiregional (Germany, Japan, Poland, U.K., U.S.) smoking cessation study in healthy smokers willing to quit. Agreement was assessed by Cohen’s weighted kappa coefficient for scale items. Intra-class correlation (ICC) was calculated for the global score. Additional stratification analyses were undertaken by gender, age, income, education, country, body mass index, and days between assessment at Visit 1 and 2. Results: 1,184 subjects (50.1% male) were enrolled. The results showed high agreement between screening and baseline visits, especially for the item on smoking behavior. Conclusions: The Lifestyle Questionnaire is a reliable measure for clinical and epidemiological studies characterizing potential behavioral confounders. The Lifestyle Questionnaire, together with existing translations in six languages, is distributed on PROQOLID™.