Background: Heat-related injury (HRI) remains a significant public health concern in heat exposed outdoor occupational groups and especially in agricultural workers like tree fruit harvesters. HRI detection is currently training and resource intensive, leading to a potential delay in diagnosis and an increasing severity at detection. Early accurate detection of HRI increases preventive and protective planning options increasing occupational safety. Urinary 8-OHdG, a biomarker of oxidative DNA damage, has shown early promise in detecting early heat health effects and acclimatization status. Objective: This study aimed to assess the practical usefulness of urinary 8-OHdG to detect early heat health effects and to characterize the relationship between urinary 8-OHdG, heat stress, heat strain, and acclimatization in outdoor tree fruit harvesters in Yakima Valley, Washington. Methods: A secondary analysis was performed on cross-sectional data collected during August and September of 2015 on 46 pear and apple harvesters from six orchards in Yakima Valley, Washington during a single work shift. The relationship between change in urinary 8-OHdG cross a work shift, heat stress, heat strain, and acclimatization were assessed using the time-weighted average Wet Bulb Globe Temperature (TWA WBGT), the Physiologic Strain Index (PSI), a validated audio computer-assisted self-interview (A-CASI), and linear regression modeling techniques. Results: There was a statistically significant mean increase in urinary 8-OHdG of 3.27 ng/dL after a work shift within participants (p-value = 1.303E-6). With greater acclimatization a lower change in cross shift urinary 8-OHdG was seen in all models, but the association was not statistically significant (p-value = 0.220). Tree fruit harvesters with higher heat stress (TWA WBGT) showed higher signs of heat strain (PSI) in the crude model (p-value = 0.025). This association was attenuated after adjusting for age (p-value = 0.052). There was no evidence of effect modification by cross shift change in urinary 8-OHdG or acclimatization on the relationship between heat strain (PSI) and heat stress (TWA WBGT) (p-values of interaction term 0.217 and 0.799 respectively). Exploratory analysis showed statistically significant associations between heat strain and acclimatization as well as with a cross shift change in urinary 8-OHdG and heat stress (p-values 0.038 and 0.019). Conclusions: Yakima valley tree fruit harvesters are working in heat exposed conditions and are at risk for heat-related injury. Urinary 8-OHdG shows promise as a biomarker of heat exposure: it is able to be successfully measured in field conditions, found to be elevated after a heat-exposed work shift, found to be less elevated with acclimatized heat exposure, found not to be associated with heat strain, and found to be associated with heat stress. The full potential of 8-OHdG to detect early heat health effects requires further study which the results of our study strongly support. Greater heat stress may be associated with greater heat strain, but a more definitive conclusion requires larger studies. Defining and understanding the full relationship between heat stress, heat strain, acclimatization, and urinary 8-OHdG is complex and more research is needed.