Background: Alzheimer-associated neuronal thread protein (AD7c-NTP) has been found to be a candidate biomarker of Alzheimer's disease (AD). Objective: To investigate the effects of urine collected time, different preservatives addition, and storage condition on the measurement of urine AD7c-NTP by enzyme-linked immunosorbent assay (ELISA). Methods: Three hundred urine samples were collected from 20 participants at three time points on five consecutive days. These samples were immediately placed at 4 degrees C and detected within 2 h. The single spot samples of the first day morning were split into eleven duplicate aliquots (a-k) of 1 ml each, (a) without any preservative (untreated), (b) containing boric acid (2 g/L), (c) containing NaHCO3 (5 g/L), (a-c) were detected at six different time points. For the other eight preservative-free samples, (d-g) were stored at -20 degrees C and (h-k) were stored at -70 degrees C, respectively, detected at different time points. All of the results were compared with the baseline urine. Results: The urine AD7c-NTP levels at different time points behaved stably (p > 0.05). Urine samples without any preservative increased over time, and compared with the NaHCO3 addition group, boric acid addition group behaved stably. Samples stored at -20 degrees C and -70 degrees C led to an obviously false positive. Conclusions: AD7c-NTP can be tested using random urine instead of the first morning urine. If the specimen cannot be tested in time, boric acid appears to be an acceptable preservative with storage at 4 degrees C, freezing is not recommended.