Estimation of physical activity using 24 h-accelerometry requires detection of accelerometer non-wear time (NWT). It is common practice to define NWT as periods >60 minutes of consecutive zero-accelerations, but this algorithm was originally developed for waking hours only and its applicability to 24 h-accelerometry is unclear. We investigated sensitivity and specificity of different algorithms to detect NWT in 24 h-accelerometry compared to diary in 47 ActivE and 559 KORA participants. NWT was determined with algorithms >60, >90, >120, >150, or >180 minutes of consecutive zero-counts. Overall, 9.1% (ActivE) and 15.4% (KORA) of reported NWT was >60 minutes. Sensitivity and specificity were lowest for the 60-min algorithm in ActivE (0.72 and 0.00) and KORA (0.64 and 0.08), and highest for the 180-min algorithm in ActivE (0.88 and 0.92) and for the 120-min algorithm in KORA (0.76 and 0.74). Nevertheless, when applying these last two algorithms, the overlap of accelerometry with any diary based NWT minutes was around 20% only. In conclusion, only a small proportion of NWT is >60 minutes. The 60-min algorithm is less suitable for NWT detection in 24 h-accelerometry because of low sensitivity, specificity, and small overlap with reported NWT minutes. Longer algorithms perform better but detect lower proportions of reported NWT.