The study by Nair et al measured the sex-dependent effects of a chronic variable stress paradigm on neuroendocrine endpoints in male and female mice, including circulating hormone levels and hypothalamic, pituitary, and adrenal mRNA expression. The paper describes relatively modest effects of CVS on baseline hormone levels and mRNA expression in the hypothalamus and pituitary, with little effect of sex on chronic stress plasticity. They also found an effect of chronic stress on the estrous cycle and ovarian histological markers of reproductive function. The study is comprehensive and thorough, and methodologically rigorous. My only significant concern with the study design, given the modest changes observed, is with the 2-week duration of the chronic stress paradigm and whether 2 weeks is sufficient to bring out plastic changes induced by chronic stress. The purpose of the study, presumably, is to provide a reference for hormonal changes that occur with a chronic stress model that avoids habituation and that, therefore, approximates chronic stress in humans. While many studies in mice use the 2-week CVS paradigm, this becomes more of an issue when negative results are found. Then the question arises that at what point is the CVS duration sufficient, e.g., if negative results are registered with 3 weeks of CVS, is this enough? And, is that time point the same for males and females, since females appear to be more resilient to chronic stress than males and might need up to 4 weeks of CVS to show changes? These, I believe, are important questions that merit a more in-depth discussion. For example, is the conclusion we should draw based on this study that C57BL/6 mice are resilient in their neuroendocrine response to chronic variable stress, or is it more nuanced than that?

Minor comments/suggestions:

Please describe briefly how controls were handled – e.g., twice a day for X min for 14 days?

Please describe how the tail tip blood sample was taken (i.e., was the tail tip snipped off or was a cut administered to the tail) and whether it generated a Cort response, and discuss whether this may have affected subsequent measures from the blood collection 25 min later.

It is stipulated that plasma TSH was measured in duplicate, but none of the other hormones were. Is there a reason for this, e.g., not enough plasma? Surprisingly, the TSH levels, along with the LH levels, had the highest variability of the hormones tested. Are these measures consistent enough to say with confidence that there was no difference between control and CVS?

Line 231 – Adrenal weights: “No significant CVS x stress interaction was observed.” This presumably is a mistake and should be CVS x sex interaction? Thymus weights: Provide the direction of change, increase or decrease, in the thymus weight.

It’s not clear why the effects of CVS on hormone levels and on pituitary mRNA expression are grouped under one subheading. I suggest separating these into two sections to correspond to the different figures.

The lack of effect of CVS on basal stress hormones (Cort, prolactin) is somewhat surprising, given what has been reported in the literature. Is there an explanation for this (or did I miss it)?

It is not clear why there are two representative graphs of the estrous cycle presented for both controls and CVS.

There is a significant effect of time on the estrous cycle. Does the time refer to the first half vs. second half? If this refers to changes over the 5-day cycle, is it worth including, since this is expected? A clarification is requested here.

The authors have provided additional discussion and context for the use of a two-week chronic variable stress paradigm that address my concern for the interpretation of the negative findings with respect to the chronic stress plasticity of neuroendocrine systems and sex-by-stress interactions.