Cortisol secretion is regulated by circadian rhythm, which is influenced by zeitgebers like light. In India, the entire country operates under a single time zone, Indian Standard Time, which may not align with the local sunrise timing across different regions. This study aimed to compare the basal serum cortisol levels between 06:00 AM and 09:00 AM in Guwahati, Assam, where sunrise occurs earlier compared with the western part of the country. A cross-sectional pilot study was conducted from December 2022 to June 2023 in a tertiary care hospital in Guwahati. Serum cortisol samples were collected at 06:00 AM and 09:00 AM from 25 healthy adult participants once in winter and again in summer. Descriptive statistics and paired Student's t-tests were used. The mean serum cortisol levels at 06:00 AM in winter, summer and overall were 13.2, 13.4 and 13.3 μg/dL, respectively. At 09:00 AM, the mean serum cortisol levels in winter, summer and overall were 8.2, 7.7 and 8.0 μg/dL, respectively. Significant differences were observed between the 06:00 AM and 09:00 AM cortisol levels in both winter and summer (P <0.001). This study highlights the importance of considering the influence of earlier sunrise on circadian rhythm, cortisol secretion and sampling protocols. Recognising the impact of earlier sunrise on cortisol secretion and adapting sampling protocols accordingly to align with the local sunrise can provide a more accurate assessment of basal cortisol levels and help avoid potential misinterpretation and diagnostic challenges associated with low values.