The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain -isoenzyme increased significantly (P < 0.02) from 0.566 ± 0.077% in Sed rats to 0.871 ± 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca²⁺]_i levels. Compared with Sed myocytes, systolic [Ca²⁺]_i levels in HIST myocytes were higher at 0.6 mM [Ca²⁺]_o, similar at 1.8 mM [Ca²⁺]_o, and lower at 5 mM [Ca²⁺]_o. The amplitudes of [Ca²⁺]_i transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca²⁺]_i transient decline, an estimate of sarcoplasmic reticulum (SR) Ca²⁺ uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na⁺/Ca²⁺ exchanger, but SR Ca²⁺-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca²⁺]_i transient amplitudes under the conditions studied. We speculate that downregulation of Na⁺/Ca²⁺ exchanger may partly account for the decreased contractility in HIST myocytes.