BACKGROUND: It is not clear whether, in established vapers, cotinine levels remain stable or change over time. METHODS: We enrolled 98 exclusive users of e-cigarettes on websites and forums dedicated to smoking cessation and to e-cigarettes. We collected saliva vials by mail in 2013-2014 (baseline), and collected a second saliva vial eight months later (follow-up) in the same participants. Participants had not used any tobacco or nicotine medications in the previous five days. Cotinine in saliva was analyzed with liquid chromatography-mass spectrometry. Use of e-cigarettes, tobacco and nicotine medications was self-reported. RESULTS: All participants were former smokers, and 99% were using e-cigarettes daily. They had already been using e-cigarettes for nine months on average at baseline. The median cotinine level was 252ng/mL at baseline (quartiles: 124-421ng/mL) and 307ng/mL at follow-up (114-466ng/mL, W=0.9, p=0.4 for change over time). The median concentration of nicotine in refill liquids was 11mg/mL at baseline (quartiles: 6-15mg/mL) and 6mg/mL at follow-up (5-12mg/mL) (Wilcoxon signed rank test: W=5.2, p<0.001 for change over time). The median volume of e-liquid used per month was 80mL at baseline (quartiles: 50-130mL) and 100mL at follow-up (60-157mL, W=3.3, p=0.001 for change over time). CONCLUSION: In experienced e-cigarette users enrolled online, cotinine levels were similar to levels usually observed in cigarette smokers. Over time, these users decreased the concentration of nicotine in their e-liquids, but increased their consumption of e-liquid in order to maintain their cotinine levels constant.