Dialysate calcium concentration (d[Ca]) might have a cardiovascular impact in patients on haemodialysis (HD) since a higher d[Ca] determines better hemodynamic tolerability. We have assessed the influence of d[Ca] on global longitudinal strain (GLS) by two-dimensional echocardiography using speckle-tracking imaging before and in the last hour of HD. This is an observational crossover study using d[Ca] 1.75 mmol/L and 1.25 mmol/L. Ultrafiltration was the same between interventions; patients aged 44 ± 13 years (N = 19). The 1.75 mmol/L d[Ca] was associated with lighter drop of blood pressure. Post HD serum total calcium was higher with d[Ca] 1.75 than with 1.25 mmol/L (11.5 ± 0.8 vs. 9.1 ± 0.5 mg/dL, respectively, p < 0.01). In almost all segments strain values were significantly worse in the peak HD with 1.75 mmol/L d[Ca] than with 1.25 mmol/L d[Ca]. GLS decreased from -19.8 ± 3.7% at baseline to -17.3 ± 2.9% and -16.1 ± 2.6% with 1.25 d[Ca] and 1.75 d[Ca] mmol/L, respectively (p < 0.05 for both d[Ca] vs. baseline and 1.25 d[Ca] vs. 1.75 d[Ca] mmol/L). Factors associated with a worse GLS included transferrin, C-reactive protein, weight lost, and post dialysis serum total calcium. We concluded that d[Ca] of 1.75 mmol/L was associated with higher post dialysis serum calcium, which contributed to a worse ventricular performance. Whether this finding would lead to myocardial stunning needs further investigation.