This study investigated the acute physiological and performance responses to a simulated prolonged service game designed to replicate the most demanding phases of competitive tennis. Fourteen nationally ranked male players (17.8 ± 0.9 y) performed 12 consecutive maximal intensity serves, each immediately followed by eight alternating forehands and backhands. Internal load was monitored before, during and after the protocol using heart rate, oxygen uptake and venous blood samples (ammonia, creatine kinase, albumin, sodium, calcium, lactate and glucose). External load was quantified with a Doppler radar gun to measure ball speed and high-frame-rate video to assess serve precision. Statistical significance was set at p ≤ 0.05. Plasma concentrations of ammonia and creatine kinase (both p < 0.01), albumin, sodium, calcium (all p < 0.05), lactate (p < 0.01) and glucose (p < 0.05) increased from baseline to post-protocol. Heart rate and oxygen uptake rose rapidly in the initial phase and stabilized at ~85 and 62% of maximal values, respectively. Serve speed and precision began to decline from the sixth point onward, with reductions of 5.4 and 29%, coinciding with peak lactate and ammonia values. A prolonged service game imposes considerable anaerobic-metabolic, cardiovascular, and neuromuscular stress on competitive tennis players, producing measurable impairments in technical performance well before task completion. Systematic monitoring of blood metabolites and electrolytes can help coaches individualize recovery intervals and design conditioning programs that enhance players’ tolerance to repeated high-intensity serving, thereby preserving technical consistency under competitive fatigue conditions.