Longitudinal data are needed to investigate chronic disease causation and improve prevention efforts for Alaska Native and American Indian (ANAI) people. This paper describes the methods used to conduct follow-up data collection of a longitudinal cohort that enrolled ANAI adults between 2004 and 2006 in south central Alaska. The follow-up study re-examined ANAI participants in a large, urban centre in south central Alaska between 2015 and 2017. Computerized surveys were used to collect self-reported health, lifestyle, physical activity, and diet data. Clinical measurements included blood pressure, fasting blood glucose and lipid panel, urine albumin/creatinine, height, weight, and waist and hip circumference. Participants were provided individual results at the conclusion of their visit. A total of 1320 south central Alaska study participants completed the baseline visit. Study staff attempted to contact all living cohort members for inclusion in the follow-up study. More than 11,000 attempted contacts were made. Of the 637 available for participation, 388 completed the follow-up visit. The proportion of women increased from baseline to follow-up examinations (67 vs. 72%, p < 0.01). Self-reported health status of being married or living as married (46% vs. 39%, p < 0.01), and those reporting being employed or self-employed (55% vs. 47%, p < 0.01) were higher at follow-up when compared to baseline. Almost all participants at follow-up (97%) agreed to long-term storage of biological specimens for future study. Despite demographic differences between the follow-up and baseline cohorts, longitudinal data collected will provide novel insight on chronic disease development and prevention for ANAI people as well as other populations.