Background: Glyphosate-based herbicides (GBH) are widely used in transgenic agricultural crops (e.g., soybeans, corn, wheat, rice, coffee, apples and grapes), increasing nutritional, environmental, and occupational exposure to pesticides1. At high concentrations, GBH has been associated with oxidative stress, altered heat shock protein (HSP) expression, and impairment in glucose metabolism2-3. However, these effects at chronic, lower-level exposure remain poorly investigated, especially when comparing sexes. Aim: To evaluate whether chronic exposure to GBH, at a dose corresponding to the Acceptable Daily Intake (ADI) in Brazil (0.5 mg/kg/day), alters biomarkers related to the HSPs, oxidative stress, and glucose metabolism of male and female rats. Methods: Twenty-four adult Wistar rats were allocated into four groups: Control-Male (CM), GBH-Male (GBHM), Control-Female (CF), GBH-Female (GBHF), (n = 6/group; Ethics Committee Approval 028/24). Exposed groups received 0.5 mg/kg/day of GBH orally (drinking water) for 12 weeks, whereas controls received water. Water and food consumption and body weight were recorded weekly. At the end of the experimental period, a glucose tolerance test (GTT) was performed; the animals were euthanized, and the liver was collected for HSP70 analysis by Western blot, and lipid peroxidation was measured spectrophotometrically. Data were analyzed by normality test, and Student's t-test or ANOVA were applied as appropriate (or respective non-parametric tests), with P<0.05 considered significant. Data were expressed as mean ± s.d. Results: GBHM and GBHF groups ingested similar doses of GBH over 12 weeks of exposure (mg/kg/day: GBHM = 0.495 ± 0.022 vs. GBHF = 0.498 ± 0.032, p > 0.854). In males, GBH exposure increased food intake (g/day: CM = 19.6 ± 0.7 vs. GBHM = 20.3 ± 0.7, p = 0.0008), body weight (g: CM = 487 ± 12 vs. GBHM = 522 ± 34, p = 0.036) and induced alterations in glucose homeostasis, as evidenced by reduced hepatic glycogen (mg/g tissue: CM = 4.19 ± 1.05 vs. GBHM = 2.29 ± 1.57, p = 0.034) and an increased glucose intolerance observed after GTT (mg/dl/min: CM = 12720 ± 2693 vs. GBHM = 15010 ± 1786, p = 0.037). No alterations were found in HSP70 expression (p = 0.394) and lipid peroxidation levels (p = 0.385). In females, we only found an increase in liver cytoprotective HSP73 expression (A.U.: CF = 0.024 ± 0.015 vs. GBHF = 0.036 ± 0.010, p = 0.043). Conclusion: Chronic exposure to GBH at a dose corresponding to the Acceptable Daily Intake by the Brazilian regulatory agency induces sex-dependent metabolic alterations that can be related to estrogen regulation of the HSP cytoprotective response. References: 1. Xu J et al. (2019) Food Control 106, 106710. 2. Jayaraman S et al. (2023) Int J Biol Mac 242, 124917. 3. Li Y et al. (2019) Toxin Rev 40, 1071.