Introduction: During early drug development trials, electrocardiograms (ECGs) in healthy volunteers who are in a fasting state are evaluated to screen for possible adverse cardiac effects. However, the effect of the duration of fasting on electrocardiographic parameters is largely unknown. We compared the effects of fasting on standard 12-lead electrocardiographic recordings. Methods: Electrocardiograms were available for 432 healthy subjects (mean age 28.5 ± 12.5; 88.9% male) who participated in early drug development studies after 4- and 10-hr fasting. All ECGs were automatically analyzed for conduction intervals and wave amplitudes with the Marquette 12SL algorithm and compared among fasting duration. Mixed model analyses were used to identify confounding variables. Results: After 10 hr of fasting, compared to after 4 hr of fasting, mean P-wave duration and amplitude were reduced by 1.95 ± 1.48 ms and 2.18 ± 2.75 μV, mean R wave and S wave amplitude were decreased by 25.83 ± 31.16 μV and 55.39 ± 78.72 μV, mean QRS duration was decreased by 1.84 ± 6.61 ms, and mean T-wave duration and amplitude were decreased by 2.06 ± 0.72 ms and 9.36 ± 17.21 μV (lead II). The mean PR interval was prolonged by 4.26 ± 17.67 ms, the ventricular rate was reduced by 3.64 ± 8.61 min, and QTcF was reduced by 3.87 ± 14.50 ms. These observations persisted after correction for demographics, electrolytes, blood pressure, heart rate variability, and diurnal variation. Conclusion: The present analysis showed that 10-hr fasting compared to 4-hr fasting resulted in changes to the surface ECG, consisting of a reduced wave amplitude and duration and increased isoelectric interval duration.