This study was the first study to examine the three circulatory biocompartments (plasma, white blood cell layer (WBC) and red blood cell layer (RBC)) and determine PF concentrations before and after exercise. Proenkephalin peptide F (PF) is an enkephalin-containing peptide found predominantly within the adrenal medulla. PF is co-packaged with epinephrine, and both can be co-secreted in response to similar stimuli. PF and epinephrine have shown immunomodulating properties. Ten healthy resistance trained men performed six sets of 10 RM squats with 2 min rest periods between sets and 10 healthy active men were matched and served as resting controls. Blood samples were obtained pre-exercise, immediately post-exercise and 15 min post-exercise and were analyzed for lactate, cortisol, epinephrine and biocompartmentalized PF. There was no change in resting control values measured across time within respective PF biocompartments and endocrine profile, indicating stability and technique validity of peptide F across the time period measured. As expected, the acute resistance exercise protocol caused an increase in lactate at 15 min post-exercise. Circulating epinephrine increased immediately post-exercise and returned to baseline during 15 min into recovery. Plasma PF increased immediate post-exercise and 15 min post-exercise, while WBC-PF and RBC-PF only increased at 15 min into recovery. For all time points tested, resting and exercise WBC-PF and RBC-PF concentrations were lower than plasma PF thus indicating a concentration difference across the three different biocompartments within the same blood sample. The presence of PF within all three biocompartments of whole blood may indicate the potential for biological transport and interactions with other cells in other biocompartments of the blood.