Sure, Here are the travel tips for your journey:

Best Time to Visit:
For sightseeing, the best time to visit Rome and Florence is from April to June and September to October when the weather is pleasant.
Venice is best visited from September to November. For Madrid and Barcelona, the springtime March-May or fall September-November is usually the best.
Avoid the peak summer season (July and August) due to the heavy tourist crowd.

Route and Accessibility:
Consider taking a flight into Rome and out of Madrid, and travel by train in between these cities. The train service in Europe is excellent and punctual.

Altitude and Acclimatization:
Altitude is not an issue in these European cities. However, if you have arrived from a different time zone, give yourself a day or two to get over the jet lag.

Accommodation and Booking:
Booking.com, Airbnb and local hotel websites are good options to book your stays. Do remember to plan and book your accommodations in advance especially during peak tourist seasons.

Clothing and Essentials:
Pack some comfortable clothing and shoes suitable for a lot of walking as these cities are best explored on foot. Always keep a light jacket or cardigan handy as evenings can get slightly cool. Also, carry a universal adapter for your electronics.

Health and Safety:
Carry any personal medication with prescriptions. Stay hydrated and wear sun protection. Also, be aware of your belongings all the time as pickpocketing is common tourist areas.

Local Etiquette and Respect:
Respect the local culture and traditions. Dress modestly while visiting religious places. Also, try to learn a few basic words and phrases in Italian and Spanish, locals appreciate it.
In restaurants, it is polite to leave a small tip for good service.