Background: Septic shock is common in the intensive care unit (ICU). The pathophysiology is poorly understood but prolonged sympathetic activation leading to autonomic dysfunction may be involved. Pupillary light response (PLR) is a fast, inexpensive, noninvasive way to measure autonomic nervous system function. The aim of the study was to observe dilation velocity of the PLR (PLR_dil.vel.) in patients with and without septic shock and explore whether other factors influenced the possible association. We hypothesized that the presence of septic shock in intensive care patients is associated with changes in sympathetic autonomic tone, which can be observed as changes in PLR_dil.vel. Methods: In this prospective observational cohort study, we included 91 adult patients acutely admitted to a mixed ICU. The patients were followed for the development of septic shock until ICU discharge. PLR_dil.vel. was measured with a portable pupillometer two times a day. We used linear mixed models to analyze for an association between PLR_dil.vel and septic shock along with several covariables. Results: Ninety-one patients were enrolled and of these, 35 were in septic shock. Septic shock was associated with a slowed PLR_dil.vel of 0.3 mm/s (95% confidence intervals [CI −0.4; −0.2]). Conclusions: Septic shock may be associated with changes in sympathetic autonomic tone which is supported by the findings from this study that septic shock was associated with a slower dilation velocity in the pupillary light reflex. Further studies should examine if the pupillary dilation velocity may serve as surrogate marker for changes in sympathetic autonomic nervous system activity in intensive care patients in septic shock. If so, future interventional studies should test if use of the pupillary dilation velocity may be used for earlier detection of septic shock, which could mean earlier institution of treatment measures for this condition.