PURPOSE: This study aimed to establish the diagnostic accuracy of a previously validated sleep staging system in patients with probable isolated REM sleep behavior disorder (iRBD), and to compare physicians diagnoses of iRBD based on REM sleep without atonia (RSWA) to non-REM hypertonia (NRH), a sleep measure independently associated with Parkinsonian spectrum disorders. PATIENTS AND METHODS: Twenty-six patients with a history of dream enactment behavior underwent a diagnostic PSG with simultaneous Sleep Profiler (SP) acquisition at two sites. PSG and SP records were sleep staged, and two sleep neurologists independently diagnosed iRBD based on the presence or absence of polysomnographic identified RSWA. Comparisons for PSG vs SP sleep staging and the qualitative presence or absence of PSG-based RSWA vs automated SP-detected NRH was performed using kappa coefficients (k), positive and negative percent agreements (PPA and NPA), and chi-square tests. RESULTS: The kappa scores from Sites-1 and -2 for PSG vs SP staging were different for Wake (k=0.82 vs 0.65), N2 (k=0.63 vs 0.72) and REM (k=0.83 vs.0.72). The by-site kappa values for stage N3 increased from 0.72 and 0.37 to 0.88 and 0.74 after PSG records were reedited. The kappa values for between-physician agreement in iRBD diagnoses were fair (k = 0.22). The agreement between each physicians iRBD diagnoses and NRH were also fair (k=0.29 and 0.22). Abnormal NRH agreed with at least one physicians iRBD diagnosis in 83% of the records. The PPA resulting from between-physician iRBD agreement was stronger and the NPA weaker than the values obtained from comparison of each physicians iRBD diagnosis and abnormal NRH. CONCLUSION: The potential utility of RSWA and stage N3 as neurodegenerative disorder biomarkers was influenced by between-site variability in visual scoring. The degree to which NRH was associated with iRBD was similar to the between-physician agreement in their diagnosis of iRBD using RSWA.