Previous studies have reported a relationship between stress and brain activity, and stress has been quantitatively evaluated using near-infrared spectroscopy (NIRS). In the present study, we examined whether a relationship exists between salivary amylase levels and brain activity during the trail-making test (TMT) using mobile NIRS. This study aimed to assess stress levels by using mobile NIRS. Salivary amylase was measured with a salivary amylase monitor, and hemoglobin concentration was measured using Neu's HOT-2000. Measurements were taken four times for each subject, and the values at each measurement were evaluated. Changes in the values at the first-second, second-third, and third-fourth measurements were also analyzed. Results showed that the value of the fluctuations has a higher correlation than the comparison of point values. These results suggest that the accuracy of stress assessment by NIRS can be improved by using variability and time-series data compared with stress assessment using NIRS at a single time point.