Healthy volunteers are crucial for biomedical research. Inadvertent inclusion of subjects with nonalcoholic fatty liver disease (NAFLD) as controls can compromise study validity and subject safety. Given the rising prevalence of NAFLD in the general population, we sought to identify its prevalence and potential impact in volunteers for clinical trials. We conducted a cross‐sectional study of subjects who were classified as healthy volunteers between 2011 and 2015 and had no known liver disease. Subjects were classified as presumed NAFLD (pNF; alanine aminotransferase [ALT] level ≥ 20 for women or ≥ 31 for men and body mass index [BMI] > 25 kg/m²), healthy non‐NAFLD controls (normal ALT and BMI), or indeterminate. A total of 3160 subjects participated as healthy volunteers in 149 clinical trials (1‐29 trials per subject); 1732 of these subjects (55%) had a BMI > 25 kg/m² and 1382 (44%) had abnormal ALT. pNF was present in 881 subjects (27.9%), and these subjects were older than healthy control subjects and had higher triglycerides, low‐density lipoprotein cholesterol, and HbA1c and lower high‐density lipoprotein cholesterol (P < 0.001 for all). The 149 trials included 101 non‐interventional, 33 interventional, and 15 vaccine trials. The impact on study validity of recruiting NAFLD subjects as controls was estimated as likely, probable, and unlikely in 10, 41, and 98 trials, respectively. The proportion of pNF subjects (28%‐29%) did not differ by impact. Only 14% of trials used both BMI and ALT for screening. ALT cutoffs for screening were based on local reference values. Grade 3‐4 ALT elevations during the study period were rare but more common in pNF subjects than in healthy control subjects (4 versus 1). Conclusion: NAFLD is common and often overlooked in volunteers for clinical trials, despite its potential impact on subject safety and validity of study findings. Increased awareness of NAFLD prevalence and stricter ALT cutoffs may ameliorate this problem. (Hepatology 2017;66:825–833)