Background: In low- and middle-income countries, health workers use pulse oximeters for intermittent spot measurements of SpO2. However, the accuracy and reliability when used for spot measurements has not been determined. We evaluated the repeatability of spot measurements, and the ideal observation time of measurement to guide recommendations during spot check measurements. Methods: Two one-minute measurements were done for the 3,903 subjects enrolled in the study, collecting 1Hz SpO2 and signal quality index (SQI) data. The repeatability between the two measurements was assessed using an intraclass correlation coefficient (ICC), calculated using a median of all seconds of non-zero SpO2 values for each recording (any quality, Q1), and again with a quality filter only using seconds with SQI ≥ 90% (good quality, Q2). The ICC was also calculated for both these conditions using subsets of the minute, in increasing increments of 5 seconds, up to the whole minute. Lastly, the whole minute ICC was calculated with good quality (Q2), including only records where both measurements had a mean SQI > 70% (Q3). Findings: The repeatability ICC with condition Q1 was 0.591 (95% confidence interval (CI) = 0.570, 0.611). Using only the first 5 seconds of each measurement reduced the repeatability to 0.200 (95% CI = 0.169, 0.230). Filtering with Q2, the whole minute ICC was 0.855 (95% CI = 0.847, 0.864). The ICC did not improve beyond the first 35 seconds. For Q3, the repeatability rose to 0.908 (95% CI = 0.901, 0.914). Conclusions: Training guidelines must emphasize the importance of signal quality and duration of measurement, targeting a minimum of 35 seconds of adequate-quality, stable data. In addition, the design of new devices should incorporate user prompts and force quality checks to encourage more accurate pulse oximetry measurement.