We employed a glycogen depleting session of exercise followed by a low carbohydrate diet (CHO) to investigate the modifications that occur in muscle sarcoplasmic reticulum (SR) Ca²⁺-cycling properties compared to a low CHO diet alone. SR properties were assessed in 9 untrained males [peak aerobic power (VO_2peak) = 43.6±2.6 (SE) ml.kg^-1.min^-1] during prolonged cycle exercise to fatigue performed at ~ 58% VO_2peak following both 4 days of low CHO (Lo CHO) and following glycogen depleting exercise plus 4 days of Low CHO (Ex+Lo CHO). Compared to LO CHO, Ex+Lo CHO resulted in a 12% lower (P<0.05) resting maximal Ca²⁺-ATPase activity (V_max=174±12 vs 153±10 µmol.g prot^-1.min^-1), and smaller reduction in V_max induced during exercise. A similar effect was observed for Ca²⁺-uptake. The Hill coefficient, defined a slope of the relationship between [Ca²⁺]_f and Ca²⁺-ATPase activity, was higher (P<0.05) at rest (2.07±0.15 vs 1.90±0.10) with Ex+Lo CHO, an effect which persisted throughout the exercise. The coupling ratio, defined as the ratio of Ca²⁺-uptake to V_max, was between 23 and 30% elevated (P<0.05) at rest and during the first 60 min of exercise with Ex+Lo CHO. The ~27 and 34% reduction (P<0.05) in Phase 1 and Phase 2 Ca²⁺-release, respectively, observed during exercise with Lo CHO was not altered by Ex+Lo CHO. These results indicate that when prolonged exercise precedes a short term Lo CHO diet, Ca²⁺-sequestration properties and efficiency are improved compared to Lo CHO alone.