The vulnerability of reference locations to climate and land-use changes and the need to protect reference locations is recognized as an emerging issue for bioassessment. Although reference sites are often located in remote, less developed regions, they still may be vulnerable to anthropogenic influences such as climate and land-use changes. We used several climate scenarios along with land-use projections consistent with well-developed future emissions scenarios to investigate vulnerabilities. Land-use projections were based on demographic modeling to allocate housing to 2100. Climate change scenarios produced altered thermal and hydrological regimes across the landscape; several urbanization scenarios increased development in currently rural or ex-urban areas. We examined the combined effects of these interacting changes on reference sites to determine regional vulnerabilities. Our results provide insights into the proportion of reference sites that may be vulnerable and require additional protection. The results also may guide the selection of sentinel sites for long-term monitoring and the locations of additional reference sites. The shifting baseline of present and future reference conditions requires additional understanding of future vulnerabilities to anthropogenic stressors and further research into projected shifts in biological communities.