In remote areas, visiting a laboratory for sleep testing is inconvenient. We, therefore, developed a Mobile Sleep Lab in a bus powered by fuel cells with two sleep measurement chambers. As the environment in the bus could affect sleep, we examined whether sleep testing in the Mobile Sleep Lab was as feasible as in a conventional sleep laboratory (Human Sleep Lab). We tested 15 healthy adults for four nights using polysomnography (the first two nights at the Human Sleep Lab or Mobile Sleep Lab with a switch to the other facility for the next two nights). Sleep variables of the four measurements were used to assess the discrepancy of different places or different nights. No significant differences were found between the laboratories other than the percentage of total sleep time in stage N3. Next, we analyzed the intraclass correlation coefficient to evaluate the test-retest reliability. The intraclass correlation coefficient between these two measurements: the Human Sleep Lab and Mobile Sleep Lab showed similar reliability for the same sleep variables. The intraclass correlation coefficient revealed that several sleep indexes, such as total sleep time, sleep efficiency, wake after sleep onset, percentage of stage N1, and stage R latency, showed poor reliabilities (<0.5) based on Koo and Li's criteria. In contrast, the percentage of stage N3 showed moderate (0.5-0.75) or good (0.75-0.9) reliabilities. As almost all sleep variables showed no difference and same level of test-retest reliability between the Mobile Sleep Lab and Human Sleep Lab, the Mobile Sleep Lab might be suitable for conducting polysomnography as a conventional sleep laboratory. The reduction in N3 in the Mobile Sleep Lab should be scrutinized in the larger sample, including sleep disorders. Practical application of the Mobile Sleep Lab can transform sleep medicine in remote areas.