Quantifying the mechanical behavior of skin has been foundational in applications of cosmetics, surgical techniques, forensic science, and protective clothing development. However, previous puncture studies have lacked consistent and physiological boundary conditions of skin. To determine natural skin tension, excision of in situ porcine skin resulted in significantly different diameter reduction (shrinkage) in leg (19.5 %) and abdominal skin (38.4 %) compared to flank skin (28.5 %) (p = 0.047). To examine effects of initial tension and pre-conditioning, five conditions of initial tension (as percentage of diameter increase) and pre-conditioning were tested in quasistatic puncture with a 5 mm spherical impactor using an electrohydraulic load frame and custom clamping apparatus. Samples with less than 5 % initial tension resulted in significantly greater (p = 0.011) force at failure (279.2 N) compared to samples with greater than 25 % initial tension (195.1 N). Eight pre-conditioning cycles of 15 mm displacement reduced hysteresis by 45 %. The coefficient of variance was substantially reduced for force, force normalized by cutis thickness, displacement, stiffness, and strain energy up to 46 %. Pre-conditioned samples at physiological initial tension (14–25 %) resulted in significantly greater (p = 0.03) normalized forces at failure (278.3 N/mm) compared to non-conditioned samples of the same initial tension (234.4 N/mm). Pre-conditioned samples with 14–25 % initial tension, representing physiological boundary conditions, resulted in the most appropriate failure thresholds with the least variation. For in vitro puncture studies, the magnitude of applied initial tension should be defined based on anatomical location, through a shrinkage experimentation, to match natural tension of skin. Characterizing the biological behavior and tolerances of skin may be utilized in finite element models to aid in protective clothing development and forensic science analyses.