Importance Prehabilitation programs are increasingly recognized for their potential to improve surgical outcomes. However, their efficacy remains debated, largely due to a lack of pathophysiologically driven implementation and limited personalization. Objective To determine the impact of personalized vs standard prehabilitation on preoperative physical, cognitive, and immune function and postoperative outcomes. Design, Setting, and Participants In this prospective, single-blinded, randomized interventional trial conducted from June 2020 to September 2022 in a single academic medical center, 58 patients undergoing major elective surgery were randomized to standard (n = 30) or personalized prehabilitation (n = 28) using block randomization. Those with contraindication to exercise, an American Society of Anesthesiologists score 4 or higher, in palliative care, less than 14 days between screening and surgery were excluded. Data were analyzed from April 2023 to May 2025. Intervention The personalized group received 2 weekly one-on-one remote coaching sessions tailored to individual progress in 4 domains (physical activity, nutrition, cognitive training, and mindfulness), whereas the standard group followed a paper-based program, including the same domains, without individualized support. Main Outcomes and Measures Primary clinical outcomes included cognitive assessments and physical performance measures, including the wall squat test, timed-up-and-go test, and 6-minute walk test (6MWT). The primary immunological outcomes included major innate and adaptive immune cell frequencies and intracellular signaling responses measured using a 47-plex mass cytometry immunoassay. Results Of 58 patients (median [IQR] age, 57 [45-67] years; 31 [57%] female) enrolled, 54 completed the study (n = 27 per group). The personalized group exhibited significant improvements in physical measures (eg, median [IQR] 6MWT: 496 [340-619] minutes before prehab versus 546 [350-728] minutes after; P = .03) and fewer moderate-to-severe postoperative complications (4 vs 11 Clavien-Dindo grade &gt;1; P = .04). Multivariable modeling identified profound and cell-type specific immune alterations postprehabilitation compared to baseline (area under the receiver operating characteristic curve [AUROC], 0.88; 0.79-0.97; P &lt; .001; leave-one-out cross-validation), including dampened phosphorylated protein kinase R-like endoplasmic reticulum kinase 1/2 signaling in classical monocytes and myeloid-derived suppressor cells after interleukin 2, 4, and 6 stimulation, and reduced phosphorylated cyclic adenosine monophosphate response–element binding protein signaling in Th1 cells. In contrast, the standard group showed only moderate clinical improvements and no immune changes (AUROC = 0.63; 95% CI, 0.48-0.78; P = .12). Conclusions and Relevance In this study, personalized prehabilitation significantly altered the immunome before surgery, dampening inflammatory signaling responses previously implicated in the pathophysiology of key surgical outcomes, including surgical site infections and postoperative neurocognitive decline. These changes were accompanied by improved physical and cognitive function before surgery and decreased postoperative complications. These findings support the use of personalized prehabilitation and provide an avenue for biologically driven monitoring of prehabilitation efficacy, and individual tailoring of programs to optimize surgical readiness and recovery. Trial Registration ClinicalTrials.gov Identifier: NCT04498208